Introduction
Biotechnology is a fast-growing field that offers innovative solutions for various health challenges. The AAPS National Biotechnology Conference (NBC) 2023 displayed the latest breakthroughs and innovations in biotechnology across different modalities, platforms, and therapeutic areas. The conference had two main tracks, track1, Advances in Therapeutic Development Across Modalities and track 2 Turbocharging Innovation in CMC: Driving Back to Rational Drug Design. Both tracks covered topics such as gene therapy, cell therapy, vaccine development, biologics design, biomarkers, bioanalysis and CMC challenges [1]. The conference also enabled networking, poster presentations, and free access to INTERPHEX, a leading event for pharmaceutical and biotechnology professionals. The conference presented the recent advances in gene therapy, including biodistribution, pharmacodynamics, cellular immunogenicity, and regulatory guidance. The conference also discussed the evolution of cellular therapy, including exposure monitoring, immunophenotyping, and allogeneic versus autologous therapies. Moreover, the conference highlighted the innovation based on traditional approaches, such as protein-small molecule hybrids, bi- and tri-specifics, oligonucleotides, and vaccine-based therapeutics. Furthermore, the conference explored the innovation based on non-traditional platforms, such as cutting-edge drug design and analytical tools for novel modalities, AI and machine learning applications, and breakthroughs in vaccine approaches. Additionally, the conference showcased the next generation gene therapies, such as gene editing modalities for cancer, immune, and infectious diseases.
This meeting report summarizes the current state and future directions of biotechnology research and development as presented during the conference. We hope that this report serves as a useful reference for the AAPS members who could not attend AAPC-NBC2023 and inspires future innovation and collaboration among pharmaceutical scientists.
1. Track1: Advances in Therapeutic Development Across Modalities (Bioanalysis)
1.1.1. Symposium Sessions
The symposium session titled Advances in Therapeutic Development Across Modalities Evolution of Cellular Therapies reviewed the considerations for preclinical development that included efforts supporting discovery to early-stage process development and support of preclinical studies through the opening prologue (Jawa V, BMS). Similarly associated bioanalytical strategy and readout of unique analytes that supported PKPD models were also elaborated to point out the differences from conventional PKPD of protein-based models. This session also highlighted the next generation of cellular therapies beyond autologous T cells to use of genetically modified (gd) CAR-T cells. The innovative CAR-T design/engineering even though intended to improve dose -exposure response, can also add more complexity to such constructs. Current experience and learnings of cell therapies in hematological malignancies has led to next generation of cell therapies to treat solid tumors and non-oncology disease indications [2-4].
The successful approval of CART-T cell-based therapies and their success in treating hematological malignancies as well as initial trials in solid tumors has been a “paradigm shift” for the field of oncology [5]. The next generation of the cell therapies include dual targeting chimeric antigen receptor targets to overcome resistance as well as engineered domains to enhance the T-cell cytotoxicity. Additionally, to reduce the long “vein-to-vein” times critical for cancer patients with poor prognosis, “off the shelf” allogeneic CAR-Ts with already validated targets have emerged.
The concept of PK for a “living biologic” like CART is unique both in the way it is measured through quantitation of the vector genomes using PCR and flow-based assays as well as their unique kinetics. The principles of ADME as it relates to small and large molecule do not apply[4]. The cellular kinetics from qPCR & Flow may not always be concordant within a CAR-T molecule as well as across various CAR-Ts due to : a) quantitation of multiple transgenes inserted/integrated per cell leading to higher sensitivity of qPCR than flow) b) intracellular trapping or downregulation of CAR expression what flow may not capture c) qPCR based expression may be reported as # copies/mL or # copies/mg of genomic DNA in blood leading to discrepancy in quantitation[6]. The characterization of “PK parameters” for CAR-T can be includes the initial cellular expansion parameters: Cmax, Tmax and AUC followed by cellular persistence parameters: Half-life, Clast and Tlast with the engineered co-stimulatory domain playing a key role in persistence. Some key observations related to clinical response associated with exposures from the currently approved CAR-T therapies (KYMRIAH® and ABECMA®) were as follows: a) responders typically have higher expansion (higher Cmax & AUC) vs. non-responders b) non-responders typically have delayed expansion (delayed Tmax) & shorter persistence (shorter T1/2) [7]. Higher exposures also showed a significant association of higher peak of CD19 CAR-T cell expansion with minimum residual disease (MRD)- negative Status.
A good measure of CAR-T efficacy and safety is the associated cytokine induction as noted by following observations; Efficacy; Response to YESCARTA® (Anti CD-19 CAR-T) was associated with higher peak levels of certain cytokines (IL-15 and IL-10); Safety; cytokine release syndrome (CRS), resulting from a large, rapid release of cytokines into the blood following treatment, is an on-target safety concern for all CAR-Ts [8, 9]. Treatment and management of CRS has been achieved through use of IL-6 receptor antagonist ACTEMRA® (tocilizumab) as a mitigation strategy [10]. The exposure-response had a direct relation to safety with a higher peak expansion (Cmax) associated with higher grades of CRS (the primary safety concern for CAR-Ts), other determinants being higher dose and tumor burden. Both CD4+ and CD8+ T cells were able to achieve a dose exposure response with significant impact of patient tumor burden on exposure[11]. Interestingly, tumor burden was a driver even without controlled CD4+:CD8+ dose ratio. Some patient intrinsic factors like body weight, race, gender did not impact exposure. Similarly extrinsic factors like tocilizumab treatment, number of lines of prior therapy etc. also had no impact on expansion and persistence.
The bioanalytical strategy for CART immunogenicity requires an understanding of the ability of CAR-T and associated viral vectors and engineered domains as well as residual contaminants to stimulate innate and adaptive phase immune response. The product derived risks include non-human content associated with receptor binding extracellular domains/CDR/linkers and domain junctions in mouse /fully human scFv. These regions can be presented on MHC class I/II and activate cytotoxic T cells and T-effector cells. The TCR variability can also contribute to the risk. Hence an assessment of adaptive phase immune response through measurement of T-effector mediated antibody responses and CTL mediated cellular immune response may be needed to monitor for the abovementioned risks.
Similarly, the residual production related proteins like AAV /Lentiviral associated proteins; expansion and editing factors like expamers/ streptavidin, and CRISPR / Talen proteins can activate innate immune responses through engagement of pattern recognition receptors as well as toll-like receptors. Such residual proteins may also be associated with preexisting reactivity that can have deleterious effects. Additional risk factors are being evaluated for next generation cellular therapies like the allogenic CAR-T where graft vs host disease due to MHC mismatch and related complications can limit the efficacy. These risk factors If identified would require monitoring for innate phase immune responses.
Other factors are related to patient and disease. For oncology indications, especially with hematological malignancies where B-cells are targets and lymphodepletion protocols are instituted prior to dosing, the risk for an immune response following dosing may be low. The risk may be higher for patients with solid tumors where lymphodepletion may not be required and B-cell may be functional. Similarly, for CAR-T therapies being evaluated for managing autoimmune disease, the baseline immune reactivity and hyperimmune status of individuals can contribute to a higher risk. Most patients also have pre-existing reactivity to AAV/Cas that can limit the gene expressions post-dosing. Patients previously dosed with other CAR-Ts can also develop and increased risk to boost immune responses to a new CART due to common regions in the old and new CAR-T and impact expansion and persistence.
These patient and treatment derived factors would again need an implementation of a robust bioanalytical strategy for clinical immune monitoring.
The most significant impact of immunogenicity would be on patient safety with adverse events like injection site reactions (ISRs) and anaphylaxis as well as a loss of efficacy caused by destruction of CAR-T leading to decreased expansion and persistence. Such effects could be more pronounced during redosing. The ADA response can neutralize CAR function. In the armored CAR-Ts (chimeric autoantibody receptor), there could be potential ADA that could cross-react with endogenous antigens[12].
The risk assessments for CAR-Ts can leverage the risk assessment assays and tools that have been used for conventional biologics. These include in silico tools to evaluate MHC Class I and II binding, novel tools predicting antigen processing and presentation and tolerance and in vitro T cell proliferation assays to detect antigen specific responses to extracellular domains of CAR-T/the whole construct or to the overlapping peptides of CDRs/linkers/domain junctions. Additionally, the MAPPS assays could also provide knowledge of domains that are likely to be processed and presented in context of MHC class I and II. These designed peptides could also be used as reagents for clinical ELISpot /CTL assay and support further validation of algorithm development. Some new tools that have been developed to assess for innate responses to residual process related proteins include whole blood / PBMC / engineered TLR cell line based innate activation assay.
Some mitigation strategies that can be implemented include selecting ScFv / domain junctions / linkers with the least risk as well as optimizing/deimmunizing the CAR-T receptor construct to be close to wild type CDRs and moving of junctions. Similarly, risk from allogenic CAR-T constructs can be reduced by insertion of TCRs, and HLA I/II/CD52 deletion using CRISPR or TALEN mechanisms and expression of Siglec ligands. Similarly, the process related impurities can be minimized, and additional clinical monitoring can be instituted per patient. Further product specs can be minimized based on in vitro risk assessments. Additional understanding of mechanisms can be obtained from clinical experience of subjects especially related to humoral and cellular immune response, pre-existing antibody status, HLA of subjects, mapping of T cell epitopes and correlate it back to the predictive risk assessment outputs.
Considerations for next generation CAR-T modalities
The next generation of cell therapies need to be designed to overcome tumor antigen heterogeneity and the hostile microenvironment around the tumor. Some examples of a strategy for next generation are listed below:
Non-small cell lung cancer: target specialized proteins called drug-tolerant “persister” cells with CART and targeted agents. Most of the newer immunotherapy approaches are ineffective for small cell lung cancers with many subtypes, each with different proteins associated with cancer cell membranes. Hence novel engineered CART with specificity against different proteins may be more effective. It was noted that there was a lack of response of chemotherapy treated cells to CAR-T. Hence increasing tumor antigen derived expression on cell membrane could make better candidates for CAR-T cell treatment.
Renal cell cancer: An off the shelf allogeneic CD70 CART therapy is being developed. The patients heavily pretreated with median 3 lines of prior therapy with advanced metastatic cancers with few options were treated with the low dose of CAR-T as a one-time treatment that lasted for many months. This strategy can be applied to other solid tumors expressing CD70.
Osteosarcoma (bone cancer): The cancer-associated fibroblasts that play a major role in tumor progression and metastases were destroyed by CART leading to disruption of ECM by altering of gene expression. The modified T cells could penetrate osteosarcoma tumors through engagement of vimentin. The destruction of cancer associated fibroblasts led to changes in tumor environment leading to additional T-cell infiltration and tumor killing.
Some recent examples of development efforts of CAR-T in solid tumor and associated immunogenicity:
- Targeting α-folate receptor with metastatic ovarian cancer: antibodies (Abs) against CAR-T were observed that were associated with reduced CAR-T activity against tumor cells and may have led to the rapid clearance of CAR-T as well [13].
- Targeting tumor-associated glycoprotein (TAG-72): neutralizing antibodies against ScFv was associated with elimination of CAR-T[14].
- Targeting carbonic anhydrase IX (CAIX) on renal cancer carcinoma (RCC): • high incidence of HACA (6/7, 85.7%) compared with using mAb alone (30%), suggesting higher immunogenicity when on cell surface than in a soluble form; HACA also inhibited cytotoxic activity of CAR-T [15].
- Targeting second generation of CAR-T against solid tumor: FRP5-ScFv against HER2-positive sarcomas and glioblastoma: • CAR transgene can be detected up to 2 years; however, clinical responses were unsatisfactory • Cellular and humoral anti-CAR was not intensely investigated.
The session included examples of next generation of cell therapies ranging from CRISPR-engineered off-the shelf CAR-T and TCR-T cells evading host T and NK cell rejection (Yuanxin Xu, Intellia Therapeutics), the use of allogeneic cell therapies in transplant medicine to replace or support dysfunctional/absent cells in degenerative disease or traumatic injury (Brian M. Culley, Lineage Cell Therapeutics) and use of Unconventional γδ T cells platform in Fight Against Cancer (Kondala Atkuri, Takeda)
Potent CRISPR-Engineered Off-The-Shelf CAR-T and TCR-T Cells Evading Host T and NK Cell Rejection: Yuanxin Yu, Intellia Therapeutics
Intellia’s CRISPR-based genome editing technology can be used to engineer CAR-T and TCR-T for allogeneic ex vivo cell therapies. The talk highlighted the critical attributes for allogeneic T cell therapy to reduce graft rejection (by host T cells and NK cells) and reduce risks of graft-versus-host disease (GVHD). Examples were provided to assess T cell potency and reduce risks of graft rejection and GVHD. Current autologous cell therapies with individualized manufacturing for each patient have challenges. 1) Leukapheresis to collect patient T cells, 2) T cells are reprogrammed with patients undergoing leukodepletion therapy to help prepare for infusion of engineered T cells, and 3) Re-programmed T cells are infused and patient is monitored for successful engraftment. As personalized medicine, the autologous therapies have technical and logistic challenges.
Potential solutions to develop next-generation engineered CAR-T and T cell therapies were to move from autologous to allogeneic product using T cells from healthy subjects.
Intellia’s differentiated approach to engineering cells includes the use of proprietary LNPs to deliver genome editing tools to knockout genes, in combination with viral mediated delivery of transgenes of interest for insertion. This approach enables a better quality cell product than electroporation, yielding desired attributes for potency, and safety profiles. This approach enables a better -quality cell product with desired attributes and benefit -safety profile with minimal double stranded breaks (DSBs), random insertions and genotoxicity risks. There are three major concerns that were highlighted related to allogeneic therapies along with suggested mitigation strategies:
- GVHD where T cell receptors (TCRs) from allogeneic T cells recognize host antigens resulting in cytotoxicity to host tissues. This can be resolved by knocking out the endogenous TCR,
- Graft rejection via host T cells; this can be mitigated by confirming that host human leukocyte antigen (HLA) molecules must match between donor and recipient preventing rejection from host CD8 (via Class I) and host CD4 (via Class II) T cells. A knockout of pan HLA Class II / HLA Class I-A alleles and matching the remaining HLA Class I alleles can prevent CD4 or CD8-mediated rejections,
- Graft rejection via host natural killer (NK) cells that attack engineered cells, lacking or having low HLA class I expression, can be addressed by knocking out HLA-A but retaining HLA -B and -C that are engaged by NK cell inhibitory receptors to block NK-mediated cytotoxicity.
A simplified strategy will be established by matching HLA Class I B and C alleles between allogeneic donor and recipient preventing GVHD and graft rejections.
A bioinformatics analysis confirmed that a limited number of donors can provide broad HLA coverage for patients (such as US and EU coverage).
Intellia’s integrated approach, applicable to CAR-T and TCR-T, addresses the main concerns with the immune mediated GVHD and graft rejection by host T and NK cells. Simplified HLA matching strategy allows donor cell engraftment and avoids long-term immunosuppression. The differentiated allogeneic platform showed key characteristics that were observed in in vitro and in vivo animal models. This strategy has proven to be highly efficient for allo-CAR-T cell engineering at the bioreactor scale. The anti-tumor activity (tumor killing and regression) was comparable between allogeneic CAR-T and autologous CAR-T products.
Allogeneic Cell Therapy as Transplant Medicine: Replacing or Supporting Cells in Patients That Are Dysfunctional or Absent Due to Degenerative Disease or Traumatic Injury
Brian M. Culley, Lineage Therapeutics
This talk provided a discussion of allogeneic cell-based therapies developed through pluripotent stem cell lines (PSCs) which provide an endless supply of undifferentiated starting material for all programs. PSCs can differentiate into any of the 200+ cell types of the human body. Hence this approach avoids the need for genetic editing and the clinical significance of the target cells has been validated by evolution insofar as they are the native cells of the body. Residual pluripotent cells are undetectable post-differentiation. Other advantages of such a therapy include ready to inject formulations (no dose preparation delay), a one-time treatment where cells integrate without rejection and a scalable process for clinical and commercial use.
This approach also fulfills requirements for a successful cell therapy for the following key areas:
Control (Safety) & Reproducibility: which includes source line characterization, cell banking, versatile expansion systems, differentiation process development; culture conditions and their optimization and analytical methods for in-process controls and establishing release criteria.
Purity / Identity requires a clinically compatible post-production processing, analytical method development for process control and product release, ensuring potency through functionality and performance testing and following enhancements through genetic modification (optional), use of various expression systems etc. and lastly scalability which includes scale-up of modalities, substrates, and harvesting protocols, ensure clinical and commercial throughputs to ensure reduced cost of goods.
Case Study 1: Development of RG6501 (OpRegen®) ― Allogeneic Retinal Pigment Epithelial (RPE) cell transplant to treat dry Age-related macular degeneration (AMD) with Geographical Atrophy (GA).
RG6501 (OpRegen), a suspension of human allogeneic retinal pigmented epithelial (RPE) cells currently in development for the treatment of GA secondary to dry AMD, is being developed under an exclusive worldwide collaboration between Lineage, Roche, and Genentech, a member of the Roche Group. OpRegen subretinal delivery has the potential to counteract RPE cell loss in areas of GA lesions by supporting retinal cell health and improving retinal structure and function.
Dry AMD involves the loss of retinal pigmented epithelium (RPE cells), creating an area of GA causing impaired vision and blindness. OpRegen is a one-time injection of fully differentiated RPE cells intended to: 1) replace and restore retinal tissue (anatomy), and 2) preserve or improve vision (function). A Phase 1/2a multi-center, dose-escalation trial evaluating a single administration of OpRegen delivered subretinally in patients with bilateral GA secondary to dry-form AMD has been completed and long-term follow-up is ongoing (ClinicalTrials.gov Identifier: NCT02286089). Published results from this study support the potential for OpRegen to slow, stop, or reverse disease progression in geographic atrophy secondary to dry-AMD. Twenty-four patients were enrolled into 4 cohorts in this study. The first 3 cohorts enrolled only legally blind patients with a best corrected visual acuity (BCVA) of 20/200 or worse. The fourth cohort enrolled 12 patients with impaired vision (BCVA from 20/65 to 20/250 with smaller mean areas of GA).
Preliminary evidence of outer retinal structure and visual function improvements with OpRegen was observed in Cohort 4 patients with GA and impaired vision (n=12), where patients exhibited an average 7.6 letter gain in visual acuity at 12 months in the study eye and three patients exhibited a 15 letter or greater gain in visual acuity at 12 months in the study eye. Long term vision preservation with outer retinal structure improvement observed in the OpRegen treated eye persisted for up to 4 years of follow-up.
In patients with extensive OpRegen bleb coverage, maintenance or improvement of outer retina structure was observed in treated eyes compared to worsening in fellow eyes. Five patients in Cohort 4 who had a surgically delivered bleb containing OpRegen that extensively covered their atrophic areas and the foveal center, experienced an average 12.8 letter gain in their study eye.
Slower rates of RPE and external limiting membrane (ELM) loss were observed in OpRegen treated eyes compared to fellow untreated eyes. Patients with extensive OpRegen bleb coverage of the atrophic area had maintenance or improvement of RPE and ELM layers compared to patients with limited OpRegen bleb coverage. Resolution of complete RPE and outer retinal atrophy (cRORA) near borders of baseline GA were also observed in cases with extensive coverage.
This study represents the only 5 known clinical cases of outer retinal structure improvement in dry AMD using a cell therapy transplant approach . RG6501 (OpRegen) is currently being evaluated in a Phase 2a clinical study in patients with GA secondary to AMD being conducted by Roche/Genentech (ClinicalTrials.gov Identifier: NCT05626114)..
Case Study 2: OPC1 Oligodendrocyte Cell Transplants for Spinal Cord Injuries
A primary feature of Spinal Cord Injury (SCI) is loss of mobility. The goal of OPC1 therapy is to restore arm, hand, and finger function. This increases independence and quality of life. Gains in motor function, particularly in the upper extremities, can provide significant benefits in self-care and lower costs of care. Transplantation of oligodendrocytes may provide additional upper extremity function and improve quality of life. Oligodendrocyte progenitor cells (OPCs) are precursors to the myelinating cells of the central nervous system. These cells provide insulation to nerve axons in the form of a myelin sheath which is essential for proper function of neurons. OPC1 was generated from an NIH-registered cell line as an allogeneic (“off the shelf”) cell therapy transplant. OPC1 is a one-time injection into the spinal cord with dosing to occur 3-6 weeks post-injury, providing time for consent and transportation.
Success has been reported in transplanting OPC1 directly to the site of damage in two separate clinical studies, in both thoracic and cervical SCI. Clinical experience with OPC1 thoracic clinical Trials showed no unexpected serious adverse events attributable to the OPC1 transplant: specifically, no evidence of neurological decline and no enlarging masses or further spinal cord damage and no syrinx formation was noted in 5 subjects followed for 10 years. Similarly, for the cervical phase 1/2a clinical trial (N=25) — All subjects evaluated for at least 2 years with no unexpected serious adverse events related to the OPC1 transplant and no enrolled patients displayed worsening of neurological function[16]. Durable motor improvements were observed in majority of subjects with at least 2 motor levels of improvement by 24 months and at least 3 motor levels of improvement by 36 months.
Innovations for the OPC1 clinical program include improvements in purity of cell manufacturing, and an enhanced spinal cord delivery method. Next steps for the program include a planned return to the clinic with a safety study in subacute and chronic SCI patients to test a novel delivery device, in both subacute and chronic patients.
γδ T Cells – An Unconventional T Cell based therapy platform in fight against cancer
Kondala R Atkuri, Takeda
Current cell therapies have proved to be promising and have mainly relied on ab autologous T cells to treat both hematological and solid malignancies. While CAR-T cells paved the way, allogeneic platforms that involve NK cells and γδ T cells are increasingly becoming attractive platforms for cell therapies. Dr Atkuri’s talk covered the basic biology of γδ T cells and the characteristics that make them an attractive platform for allogeneic cell therapy in cancer. A brief comparison of γδ T cells with NK and autologous T cells was also performed. The analytical activities that are needed to meet the regulatory requirements and maximize clinical understanding of the product/platform with special emphasis on cellular kinetics, immunogenicity, and other disease measurement were also presented as part of the talk.
The γδ T cells intersect the innate and adaptive immune responses with key features like fast acting response activated by distressed or infected self, potent and ability to recognize patterns and specific antigens and develop a memory response. The anti-tumor activities of γδ T cells are mediated by innate receptors and NCRs, release of cytolytic granules containing perforin and granzymes A and B, induction of Th1 cytokines including IFN-g and TNF-a, death receptor ligands that induce apoptosis and immune orchestration. Few reports from late-stage human cancers have reported pro-tumor and pro-metastatic activities largely driven by IL-17, Gal-1, gal-9 and PMN-MDSC[17].
Subset of gd cells
Vδ1 cells:
- Second most predominant subset in blood (up to 1/3 of circulating γδ T cells)
- Highly enriched in epithelial tissues including dermis, gut epithelium, spleen and liver
- Contribute most to tissue homeostasis
Vδ2 cells:
- Earliest γδ T cells to be generated
- Predominant subset in blood (70%-90%)
- Paired with Vγ9
- Used in most γδ T-cell therapies
Vδ3 cells:
- Predominant subset in liver and intestines
- Exhibits CD1d restriction
- Minor population in blood
Vδ5 cells:
Two main platforms for γδ T cells:
Vδ1: Diverse TCR1 that can respond to a variety of antigens, viruses (CMV2 and EBV) and non-peptide lipids. They can be rapidly expanded by TCR stimulation and cytokines.
Vγ9δ2: Semi-invariant TCR4 that respond to non-peptidic prenyl pyrophosphates (phosphoantigens). Can be activated by phospho-antigen presented by BTN3A16 and can be rapidly expanded by amino-bisphosphonates (zoledronate and pamidronate) [18-21].
Compared to conventional allogeneic cells (NK and ab Tcells) gd cells have following advantages; a MHC unrestricted TCR, no risk of GVHD, reasonable antigen specificity and memory, NKp30 and p44 and broad NCR expression, ease of epithelial tissue homing, Th1 focused cytotoxicity, lack of KIR expression, orchestrating adaptive response, low risk of CRS and resistant to exhaustion and inhibition.
Allogeneic gd cells with single donor treating multiple patients will also need a specific and complex bioanalytical strategy[22-25]. The complexity is proportional to the complexity of CAR design as there can be an increase in number of analytes and need for cell type and disease indication specific customization of readouts. The cellular kinetics assessment can be cell based (flow based) to detect CAR+ and CAR- cells, genomics based (PCR based) to detect CAR transgene and chimerism based (using NGS ) to detect donor vs recipient cells.
Cell therapies require a well thought out translational strategy that ties pre-clinical assessments with clinical response, biomarkers, and disease, patient and product characteristics.
1.1.2. Keynote speakers
Over the past decade, biological drug development has evolved significantly beyond standard monoclonal antibody therapeutics, into complex antibody-based constructs, genetic therapies that alter patient’s cells ex vivo, and even in vivo-directed gene therapies that are able to transform cells inside patients to express missing or wild type proteins. To give a broad perspective on the evolution of therapeutics across modalities, each keynote targeted a particular therapeutic modality. Priya Chockalingham discussed the disease pathology of sickle cell disease resulting from mutated hemoglobin, and then discussed the bioanalytical strategy to support a gene therapy product of CD34+ modified cells to express fetal hemoglobin which theoretically could correct the disease pathology. Bioanalysis played a key role in determining the efficacy and safety of this gene therapy; methodologies were developed to quantitate the efficiency of gene editing in the target cells, the levels of therapeutic and mutant hemoglobin, as well as biomarkers for hemolysis. Similarly, Karen Mueller providing a historical perspective on the clinical studies for Kymriah, as well as the bioanalytical methodologies used to measure the pharmacokinetics, immunogenicity, and pharmacodynamic effects of the cell therapy. The final keynote centered around innovation based on traditional platforms, Malaz Albutarif discussed the evolution in Clinical Pharmacology of models used to inform the drug development paradigm and the advancement of better computing systems and increased regulatory acceptance. He highlighted advances in quantitative systems pharmacology to better predict the behavior of advanced therapeutic modalities leading to informed decision making and ultimately resulting in enhanced patient outcomes.
1.1.3. Hot topics
In the “Intro to Biomarkers” Hot Topic session, a panel of industry and regulatory biomarker experts led a robust Q&A discussion on the fundamental understanding of biomarkers. There were three focal discussion points that came from this Hot Topic session including a robust discussion on CLIA, CDx, and IVDR applicability to biomarker assays, a discussion on appropriate regulatory guidance to follow and importance of nomenclature in this space, and lastly an important discussion on deriving the context of use (COU). The key takeaways are further summarized. The Hot Topic discussion was kicked off with a dynamic discussion with the audience and panelists on the topic of biomarker assays that are used for inclusion and exclusion of patients during enrollment of clinical trials. Especially noted where patient history or tests do not exist to demonstrate clinical utility prior to conducting the clinical study (e.g., for PEGylated molecules, no prior data demonstrating sensitivity to PEG and no linkage to clinical utility, or in the case of AAV based gene therapies, where patients with pre-existing antibodies to AAV are excluded from enrollment). Within the CLIA verbiage, it clearly states that if the in-vitro diagnostic test is used to make a medical decision about a patient, then it should fall under CLIA regulation. However, many companies are falling on clinical research exemption (IDE) to use in-house assays. Further challenges and confusion have also surfaced with the updated IVDR regulations in Europe. The robust discussion on this topic remains as a Hot Topic in the industry and emphasizes the need to continue sharing ideas to help us all move forward. During the discussion with the panelists and audience it was clear that nomenclature in the biomarker world is important and needs harmonizing across the industry. For the FDA a “biomarker qualification” means something entirely different than the analytical qualification of a biomarker assay. When filing a biomarker for regulatory qualification, the biomarker itself is qualified, not the assay, thus allowing that biomarker to be used in any drug application for that intended use that it was qualified for. To avoid confusion with this concept, there was a call to action for the industry to refer to analytical qualification or validation of biomarker assays as “Fit-For-Purpose Validation”. Another topic that was discussed extensively is the importance of the conversation around COU of the biomarker. It is the ownness of all key stakeholders to help derive the COU and oftentimes it is an iterative process. It was concluded that if the COU is not fully known, then we resort to bioanalytically characterizing the assay for what it can do currently and refine it until it meets its needs.
The “Hot Topic: Bioanalytical Challenges and Strategies for Novel Drug Modalities” session included 3 presentations discussing different aspects of RNA-LNP bioanalysis. Bioanalytical assessments of RNA-LNPs include LNP and mRNA PK, concentration of the mRNA-derived protein and immunogenicity of mRNA and LNP. Eric Tewalt focused on the branched DNA method to measure mRNA PK, discussed its advantages (i.e.: the lack of sample extraction requirement) and challenges. Due to the rigid requirements for incubation times and temperatures, the correct placement of the assay plate in the incubator is critical. Best practices for handling RNA-LNP in serum are to dispense samples, calibrators and QCs into lysis buffer as soon as possible to prevent mRNA degradation and to prepare paired calibrators and QCs. Jason DelCarpini focused on reference materials for mRNA-derived proteins and indicated that the reference material in an assay could be different from the proteins expressed in vivo. The recommendations for surrogate recombinant proteins include starting early in the expression and purification process, choosing mammalian production if possible, paying attention to the potential impact of tags (His, Fc, etc.) on the assay performance and maintaining a consistent source of the material during pre-clinical and clinical development. Ling Morgan discussed the use of free Ionizable lipid as a surrogate assessment of LNP PK by LC/MS/MS. The bioanalytical challenges of Ionizable and PEG lipid measurements include the lipid stability in different matrices as well as non-specific binding to plasticware and glassware. Because of this, it is recommended to measure the stability of both free lipid and LNP in matrix and to include stabilizers during sample collection to preserve lipid integrity. A fourth presentation by Sebastian Guelman highlighted bioanalytical strategies for adoptive T cell therapies. Two complementary assay platforms support cellular kinetics measurements: PCR-based and multi-parameter flow cytometry-based, each with its advantages and drawbacks. Cell therapy products can potentially elicit humoral and cellular immune responses upon administration. Two assay formats could be used to measure ADAs: bridging ELISA or cell-based. Cellular immunogenicity could be evaluated using flow cytometry or ELISpot and requires PBMCs isolated from the patients in a timely and consistent manner across clinical sites.
The “Hot Topic: Translational Intelligence in Omics” session focused on advances in the field of Omics. Timothy Garrett focused on the clinical translation of metabolomics in rare diseases, cancer, and infectious diseases using intelligent omics. The potential of metabolomics, especially lipidomics was explored for rare diseases and as diagnostic markers despite current challenges. For rare diseases such as Fabray lipid pathway, metabolomics/lipidomics could be a powerful tool to better understand the manifestations of the disease. Glycosphingolipid (Ga2 and Ga3) biomarkers were measured by HRMS in the renal tissues of a patient that confirmed the disease. The ratiometric analysis (ratio of Ga2 to ceramide) demonstrated 20x higher amounts of Ga2 in the patient sample. The challenges to the Dried Blood Spot (DBS) sampling and mitigation strategies for multi-omics workflows were demonstrated in the second case study. The levels of ceramides were elevated in the placental blood of pregnant patients with malaria. Intelligent DBS (iqDBS) was employed for extraction recovery and quantitative measurements of Phe and Tyr in PKU patient population as well. The spots were coated with an internal SIL label so the need for handling of internal standards at remote locations was eliminated.
Anup Madan’s presentation was focused on single-cell genomics for a better understanding of heterogeneity in the cellular population. The ineffectiveness of the therapy to kill all cancer cells could be attributed to the heterogeneity in patient samples. The limitation of current single-cell genomics and mitigation strategies were highlighted. Amplification biases could be mitigated by Unique Molecular Indices (UMI). Spatial multi-omics and single-cell genomics would help investigate the biological system as a whole and thus would enable a better understanding of biological variability. A case study to demonstrate population heterogeneity using single-cell analysis of T-cells via single-cell transcriptomics was discussed. Data integration coupled with network-based representation learning would help enable a deeper understanding of the variability of genes in single cells. The analysis would help enable development of the better gene therapy drugs limited by the low siRNA intake into cells and its effect on the efficacy of therapeutics. The analysis would enable a deeper understanding of the off and on-target cytotoxicity of the CAR-T-based drugs and thus would propel improved cell therapy drugs.
1.1.4. Rapid Fires
In the Rapid-Fire session focused upon recent advances in gene therapy, Jim Glick (Novartis) presented considerations for GT related diagnostic device development such as, device development regulations, early interface with global regulators, sound assay validation practices, use of control and placebo samples, clinical cutoff were highlighted. Sarah Al Hakeem (Bioagilytix) called for standardizing the AAV humoral immunogenicity assays in the industry in order to gain sufficient understanding of impact of pre-existing immunogenicity responses. Ayuko Ota-Setlik (Pfizer) presented recent advancements in developing alternative assay formats for ELISPOT assays which are cumbersome to implement. These alternatives included flow based intra-cellular cytokine staining assays and emerging PCR or cytokine release assays as proxy for T-cell activation. While the correlation data to ELISPOT assay results look promising, once validated, these novel assay formats can allow for much simplified execution in global trials.
Speakers in the rapid-fire session focused on the evolution of cell therapy covered an overview of CART cell technologies, and how these cell therapies have been evolving together with our understanding of tumor immunity including some recent strategies such as including expression of relevant cytokines involved in immune augmentation. Weifeng Xu (Merck) focused on use of CRISPR/Cas9 gene editing technology to develop allogeneic T-cell and CAR-T as ex vivo therapies, with a focus on mitigating the risk of graft vs host disease (GVHD). Various technological advancements were covered to enable more robust and efficient development of CAR-T therapies. One such example was from Xinyan Li (Janssen) highlighted an advancement in ability to extend blood sample stability time to 32h for clinical ELISPOT assays. This was accomplished by reducing cell resting time and increasing centrifugation time and this development has the potential to pave the way for wider use of the assay in global clinical trials allowing for valuable data generation.
In the rapid-fire session based on innovation around traditional therapeutics, a variety of new approaches were discussed including Tasneem Kazi (Genentech) who presented a modeling strategy to guide the development of a targeted bispecific antibody. She developed a model incorporating in-vitro data, to estimate key parameters required to design targeted bispecific antibodies. The preliminary PBPK model predicted that higher expression of tumor antigen and sub-nanomolar to nanomolar affinity of tumor targeting arm are important factors for optimal efficacy.
1.1.5. Key takeaways from Bioanalysis track
This track explored the latest technologies to accelerate and improve biotechnology drug development processes. It included topics such as cell and gene therapy, immunotherapy, biosimilars and biobetters, pharmacokinetics and pharmacodynamics, bioanalytical methods, regulatory science, and quality by design [1].
- The track featured speakers from academia, industry, and regulatory agencies who shared their insights and experiences on the challenges and opportunities in developing novel therapeutics across different modalities1.Some of the highlights from the track included: A keynote presentation by Dr. Peter Marks, director of the Center for Biologics Evaluation and Research at the FDA, who discussed the regulatory landscape and expectations for new modalities.
- Sessions on:
- Cell and gene therapy, where experts discussed the latest advances in vector design, manufacturing, delivery, biodistribution, immunogenicity, and safety of these promising modalities [1].
- Immunotherapy, where speakers showcased the development of novel bi- and tri-specific antibodies, CAR-T cells, and bispecific T-cell engagers for cancer treatment1.
- Biosimilars and biobetters, where speakers highlighted the challenges and strategies for demonstrating biosimilarity, interchangeability, and superiority of these products1.
- Pharmacokinetics and pharmacodynamics, where speakers presented novel approaches and methods for modeling and predicting the exposure-response relationship of new modalities1.
- Bioanalytical methods, where speakers shared their best practices and innovations for developing robust and reliable assays for new modalitieshttps://www.aapsnewsmagazine.org/meetings-jan231.
- Regulatory science, where speakers discussed the current guidance and expectations for new modalities from different regions and agencies.
- Quality by design, where speakers demonstrated how to apply QbD principles and tools to optimize the quality attributes and process parameters of new modalities.
2. Track2: Turbocharging Innovation in CMC: Driving Back to Rational Drug Design
2.1.1. Keynote speakers
The keynote speakers in track 2 focused on CMC innovations as well as emerging opportunities and challenges in the development of new delivery modalities. Dr. Mark Prausnitz from Georgia Institute of Technology discussed his novel transdermal platform of microneedle arrays in Phase II Clinical trials in West Africa. He described the benefits of this delivery approach in terms of costs and cold chain storage, as well as patient compliance from a global perspective. Dr. Michael Molony from Sangamo therapeutics went into the analytical challenges in gene editing and gene repression, as well as regulatory challenges as it affects gene editing based biotherapeutics. Dr. Nripen Shah from PassageBio focused on CMC challenges in CNS disorders for both rare and large patient groups with regards to gene editing. Process and analytical development for some serotypes and modalities are not yet established which further restricts the ease of manufacture.
2.1.2. Symposium Sessions
Biologics play a pivotal role in shaping modern medicine, continually advancing with cutting-edge innovations [26]. The symposium titled "Overcoming Critical Challenges of Evolving Biologics" commenced with an illuminating Prologue delivered by Nicole Buist from Merck & Co., Inc. Nicole's talk highlighted the dynamic and innovative nature of biological drug products, with a particular focus on the advantages of high concentration and high-volume drug products for both healthcare providers and patients. Notably, she emphasized how excipient selection and alternative suspension technologies can further enable the development of high concentration large molecule formulations. Moreover, Nicole’s presentation reviewed the diversity in modality beyond the typical monoclonal antibodies, which shared insights on the challenges of developing these therapies as combination products and raised questions about how to better develop these products. It also delved into addressing challenges pertaining to biophysical and biochemical instability in formulation strategies, while stressing the importance of high-resolution analytical characterizations without the need for sample dilution, thus igniting discussions on the need for advanced in-situ characterizations. Diving deeper into the core themes, the symposium shed light on significant topics such as the profound impact of protein-protein interactions in high concentration formulations, offering invaluable insights shared by Christopher Roberts from the University of Delaware. Additionally, Raj Suryanarayanan from the University of Minnesota presented recent research progress on the phase behavior of mannitol along with sugar in frozen storage, revealing its crucial impact on protein aggregation in lyophilized formulations, frozen solutions, and freeze/thaw processes. Advanced characterization tools such as synchrotron X-ray diffractions was introduced to explore the phase behavior in freezing process, promoting a better understanding and controlling in biopharmaceutics manufacturing. Moreover, Mehran Yazdanian from Teva Pharmaceuticals shared invaluable insights into the challenges of developing biosimilar drug products. His presentation delved into the complexity of biosimilar drug products, offering a deeper understanding of the scientific challenges associated with regulatory guidelines. The talk also highlighted the necessary activities and strategies for successful development of biosimilar drug products, complementing experts’ opinion with compelling examples and case studies. These enriching discussions provided comprehensive insights into the ever-evolving world of biologics, reaffirming their promising future and vital role in modern medicine.
2.1.3. Hot topics
Lipid nanoparticle (LNP) enabled mRNA delivery emerged as a timely and prominent topic during this conference, garnering significant interest [27, 28]. Notably, two distinguished speakers, Rich Pelt from Pfizer, Inc. and Mark Brader from Moderna, delivered captivating presentations on this critical subject. Rich shared Pfizer's CMC Strategy for mRNA-LNP Vaccines, highlighting the unprecedented challenges and regulatory considerations faced during the COVID-19 pandemic. His inspiring story centered on implementing flexible yet predictable regulatory strategies to ensure ample manufacturing capacity for COVID-19 therapeutics and vaccines, meeting the global demand effectively. Mark's seminar emphasized the intricate structure of LNPs and RNA, elucidating the need for advanced characterization techniques to gain profound insights into LNP morphological structure and the physicochemical microenvironment of the encapsulated RNA. His compelling narrative underscored the paramount importance of a comprehensive understanding of molecular-level fundamentals that underpin pharmaceutical properties. Their talks provided a comprehensive perspective on the significance of LNP-enabled mRNA delivery and the innovative strategies employed to address critical challenges, contributing significantly to advancements in the field of medicine and therapeutics.
2.1.4. Rapid Fires
A series of rapid-fire talks centered around the theme of solid-state biologics, focusing on lyophilization and spray drying processes with the goal of enhancing protein stability through rational design and fundamental understanding of stabilization mechanisms. Fengyuan Yang from Ashland, Inc. delivered an intriguing talk on his recent study that compared the effectiveness of trehalose and sucrose in preserving and stabilizing lyophilized therapeutic proteins. Through an analysis of the physicochemical properties of lyophilized cakes, his research provided valuable insights to aid formulation design when choosing the appropriate sugar. Jing Ling from Merck & Co., Inc. presented a highly captivating study aiming to evaluate how formulation conditions impact the distribution of proteins and their structure during the drying process. By delving into the molecular level, Jing's research shed light on protein-excipient interactions and miscibility using solid-state NMR. This in-depth investigation uncovered the underlying mechanisms contributing to the distinct physical stability of protein solids derived from lyophilized and spray-dried processes. These rapid-fire talks contributed to a better understanding of solid-state biologics, paving the way for improved formulations and enhanced protein stability, thus holding immense promise for the future of biopharmaceuticals.
2.1.5. Key takeaways from CMC track
The AAPS-NBC-2023 CMC track focused on the emerging landscape of non-traditional platforms for drug delivery and novel modalities, such as cell and gene therapies. Some of the key takeaways from this track are:
- Innovative technologies have been harnessed to integrate cutting-edge resources with scientific expertise in shaping the next wave of medical interventions. Examples include drug design and analytical tools for novel modalities, innovations in vaccine approaches, enhancing scope and coverage of modalities using AI and machine learning, as well as regulatory guidance and perspective on future innovations in biotechnology
- Beyond the innovation in drug discovery, pharmaceutical and biotechnology communities are embracing emerging opportunities and advancements in Chemistry, Manufacturing, and Controls (CMC) in all stages of the drug development life cycle. This track focused on the frontiers of drug and formulation design in biologics development, modern experimental, computational, intelligent analytics, regulatory perspective, and bioengineering [29].
- The next wave of novel gene editing modalities will impact other therapeutic areas such as cancer, immune, and infectious diseases. The CMC track discussed recent advances in gene therapy, such as biodistribution, pharmacodynamics, cellular immunogenicity, as well as how these fit into evolving regulatory guidance.
Future Outlook and concluding remarks.
AAPS’ mission is to be a convener for pharmaceutical scientists from across the entire spectrum of pharmaceutical discovery and development. Since the association has brought the National Biotechnology (NBC) back as an integral part of the AAPS scientific calendar in 2021, it has been our ambition to deliver a heavily innovation-focused, cutting-edge meeting with enough flexibility in its tracks and themes to deliver programming that is as close to the current scientific pulse as possible. Innovation comes in many different forms and is not limited to the discovery and development of new modalities.
Looking ahead to 2024, Track 1 is focused on Advances in Discovery, Formulation, and Delivery of New Modalities. The track focuses on aspects of the discovery and development of biologics and new modalities. Theme 1 of this track will discuss Preformulation and Formulation Approaches for Biologics and New Modalities and explore the challenges associated with preformulation and formulation development of biologics and new modalities. Theme 2 of Track 1 will cover Drug Delivery for New Modalities and Therapies and focus on different strategies and challenges involved in the design of drug delivery systems, the delivery of biologics, and manufacturing challenges in the development of novel drug delivery systems. Theme 3 of Track 1 will focus on AI/ML in Drug Discovery and Development, clearly technologies that are transforming the landscape of drug development and reducing discovery time and cost. Track 2 of our 2024 conference is centered around novel strategies to advance biotherapeutic development through the specific lenses of targeted delivery, platform technology, high-content bioanalysis, and data sciences. Theme 1 will focus on Development Strategy for Novel Targeted Therapeutic Delivery, specifically the increasing relevance of targeted drug delivery for the field of protein therapeutics and associated methods of bioanalysis. Themes 2 and 3 are strongly grounded in analytical and bioanalytical questions, reflecting our Association’s continuing strong interest, expertise, and contributions in this space. Theme 2 will explore Advances in Analytical and Bioanalytical Platform Technology – timely, as field of analytical platform technology is racing to keep pace with the emergence of new modalities such as oligonucleotides and cell and gene therapies, as well as the increasing need for more informative analytical evaluation of traditional therapeutics such as monoclonal antibodies and antibody drug conjugates. Theme 3 will cover High Content Bioanalysis and omics, a field that is still exponentially evolving, as researchers seek to apply its gains across the discovery and development continuum.
The 2024 AAPS National Biotechnology conference will be held May 13 –16 at the Hilton San Francisco Union Square. Programming submissions will be accepted until September 26, 2023.
Author affiliations:
1 Pfizer, Early Clinical Development, Precision Medicine, Cambridge, MA, USA
2 Merck & Co., Inc., Rahway, NJ 07065, United States
3 BioAgilytix Labs, Durham, NC 27713, USA
4 Genentech Inc., South San Francisco, CA 94080, USA
5 Samford University, McWhorter School of Pharmacy, Birmingham, AL, USA
6 Moderna Therapeutics, Cambridge, MA 02139, USA
7 Kojin Therapeutics, Boston, MA 02210, USA
8 Takeda, Development Center Americas, Ltd, Cambridge, MA, 02139, USA
9 American association of pharmaceutical scientists (AAPS), Arlington, VA, 22201, USA
10 Teva Pharmaceuticals, Inc., West Chester, PA 19380, USA
Corresponding author: Mohamed Hassanein, Pfizer, Early Clinical Development, Precision Medicine, Cambridge, USA, email: mohamed.hassanein@pfizer.com
Disclosure: The views expressed in this article are those of the authors and do not reflect official policies of their employers
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