# Cell Squeeze®: Driving more effective CD8 T cell activation through cytosolic antigen delivery

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## Bibliographic record

- Title: Cell Squeeze®: Driving more effective CD8 T cell activation through cytosolic antigen delivery
- Document type: Publication
- Year: 2022
- Outlet: ESMO IMMUNO-ONCOLOGY AND TECHNOLOGY
- Cells: T cells, PBMCs, Monocytes, B cells, Dendritic Cells, NK cells
- Cargos: Polymers, Protein, Peptide
- Page URL: https://portal.bio/publications/2022-cell-squeeze-driving-more-effective-cd8-t-cell-activation-through-cytosolic-antigen-d--ajkGkhEAACoAc97K
- Markdown URL: https://portal.bio/publications/2022-cell-squeeze-driving-more-effective-cd8-t-cell-activation-through-cytosolic-antigen-d--ajkGkhEAACoAc97K.md
- Original file: https://portal-bio.cdn.prismic.io/portal-bio/65ccdab29be9a5b998b5d115_SQZ_ESMO_IOTECH.July2022.pdf

## Summary

Jong Chul Park, Howard Bernstein, Scott Loughhead, Ricardo Zwirtes, Julia Jennings, Valeria Nicolini, Christian Klein, Laura Codarri Deak, Pablo Umana, Christine Trumpfheller, Armon Sharei

## Publication text

Cell Squeeze®: Driving more effective CD8 T cell activation through cytosolic antigen delivery Jong Chul Park, Howard Bernstein, Scott Loughhead, Ricardo Zwirtes, Julia Jennings, Valeria Nicolini, Christian Klein, Laura Codarri Deak, Pablo Umana, Christine Trumpfheller, Armon Sharei

### Pii: S2590-0188(22)00022-3

DOI: https://doi.org/10.1016/j.iotech.2022.100091 Reference: IOTECH 100091 To appear in: Immuno-Oncology and Technology Received Date: 27 March 2022 Revised Date: 1 July 2022 Accepted Date: 4 July 2022 Please cite this article as: Park JC, Bernstein H, Loughhead S, Zwirtes R, Jennings J, Nicolini V, Klein C, Deak LC, Umana P, Trumpfheller C, Sharei A, Cell Squeeze®: Driving more effective CD8 T cell activation through cytosolic antigen delivery, Immuno-Oncology and Technology, https://doi.org/10.1016/ j.iotech.2022.100091. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2022 The Author(s). Published by Elsevier Ltd on behalf of European Society for Medical Oncology. Title: Cell Squeeze®: Driving more effective CD8 T cell activation through cytosolic antigen delivery Authors: Jong Chul Park1, Howard Bernstein2, Scott Loughhead2, Ricardo Zwirtes2, Julia Jennings2, Valeria Nicolini3, Christian Klein3, Laura Codarri Deak3, Pablo Umana4, Christine Trumpfheller3, Armon Sharei2 1Massachusetts General Hospital, Harvard Medical School, Boston, MA USA 2SQZ Biotechnologies, Watertown, MA USA 3Roche Innovation Center, Zurich, Switzerland 4Roche Pharmaceutical Research and Early Development, Zurich, Switzerland Corresponding author Jong Chul Park, MD Massachusetts General Hospital Address: 55 Fruit St, Boston, MA 02114, USA. E-mail: jpark73@mgh.harvard.edu Phone number: 617-724-2694Journal Pre-proof Title: Cell Squeeze®: Driving more effective CD8 T cell activation through cytosolic antigen delivery1 Highlights3  Cell Squeeze® is a novel technology that relies on temporarily disrupting the cell membrane to4 deliver cargo directly into the cytosol5  By enabling direct cytosolic delivery, Cell Squeeze® enhances MHC Class I presentation of6 antigens for CD8+ T cell activation7  With rapid, cost-effective manufacturing of cell therapies, the Cell Squeeze® technology could8 potentially improve the feasibility and accessibility of cell therapies for many indications.9 Keywords11 Cell Squeeze®, Therapeutic cancer vaccine, Cell therapy, MHC Class I antigen presentation12 Abbreviations14 Activating antigen carrier (AAC), antigen (Ag), antigen-presenting cells (APCs), human papillomavirus15 (HPV), immune checkpoint inhibitor (ICI), major histocompatibility complex (MHC), peripheral blood16 mononuclear cell (PBMC), programmed death 1 (PD-1), tumor microenvironment (TME)17 26Journal Pre-proof Abstract1 Cell Squeeze® is a novel technology that relies on temporarily disrupting the cell membrane to deliver2 cargo directly into the cytosol. This approach is applicable to a broad range of cell types (peripheral blood3 mononuclear cells, red blood cells, hematopoietic stem cells, etc.) and cargos (peptides, proteins, small4 molecules, nucleic acids, and gene-editing complexes) while minimally disrupting normal cell function.5 By enabling direct cytosolic delivery, one can use this technology to dramatically enhance major6 histocompatibility complex (MHC) Class I presentation of antigens for CD8+ T cell activation – a7 longstanding challenge for the therapeutic cancer vaccine field which has generally relied on cross-8 presentation of endocytosed antigens. In addition, by coupling improved MHC-I presentation with co-9 expression of additional stimulatory factors or systemic immune modulators, one can further enhance the10 potential impact of an anti-tumor CD8 response. Pursuing a more direct cellular engineering strategy,11 which is independent of viral transduction, genetic manipulation, and expansion steps, enables <24hr12 manufacturing of autologous cell therapies. Through generation of more sophisticated, multi-functional,13 cell-based vaccines, clinical testing of this technology will elucidate its potential for impact across14 multiple tumor types.15 Background17 As immuno-oncology is better understood, it is evident that CD8+ T cell activation and infiltration into18 the tumor microenvironment correlates with the response across tumor types, however, it has been19 historically challenging to elicit this CD8+ T cell response (1). To date, therapies aimed at priming the20 CD8+ T-cell response have had limited success.21 Approved chimeric antigen receptor T-cell (CAR T-cell) therapy, an autologous CD4+ and CD8+ T cell22 product genetically modified to express CD19, has had success in certain hematological malignancies,23 however, this success has not translated into effective treatment against solid tumors as CAR-T cells may24 not be able to infiltrate the tumor microenvironment (2) or can drive fatal toxicity (3, 4). Additionally,25 delivery of the transgene into the T-cell to manufacture CAR T-cells for clinical use is complicated.26Journal Pre-proof Established methods of delivering cargo into the cell, such as viral vectors or electroporation, could be1 used to deliver immunogenic cargo, however these techniques present different challenges (Table 1).2 Viral integration of the transgene has oncogenic and mutagenic potential, limits the size of the transgene,3 and necessitates more cumbersome release testing to control for these risks. Electroporation can be used4 as an alternative to viral vectors, however, it tends to be less efficient for integration and has been shown5 to disrupt normal cell function (5). Harsh preconditioning regimens, high manufacturing costs, and6 lengthy turnaround times further contribute to the difficulties of broadly implementing current cell7 therapies.8 While immune checkpoint inhibitors (ICIs) have aimed to rescue exhausted CD8+ T-cells by inhibiting9 negative regulators of T-cell function, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4),10 programmed death 1 (PD-1), and programmed death ligand 1 (PD-L1), only a minority of patients11 respond to treatment suggesting that ICI therapy itself is not sufficient. Failure to ICI therapy is not fully12 understood, but impaired formation of antitumor T-cells (including lack of response to tumor-specific13 antigens and inability to infiltrate the tumor microenvironment (TME)) are thought to contribute (6).14 Therapeutic cancer vaccines are a potentially promising approach to improve antigen-specific immune15 responses against cancer cells. With a long list of identified neoantigens and cancer-associated viral16 antigens, the as yet unrealized benefits of a therapeutic cancer vaccine could be substantial (7). Many17 clinical trials to demonstrate cancer vaccine proof-of-concept are currently being conducted in patients18 with human papillomavirus (HPV) associated cancers. Given the viral nature of HPV-driven tumors,19 which constitutively express oncogenic E6 and E7 proteins which are not present in non-cancerous cells,20 these cancers are ideal for evaluating a therapeutic cancer vaccine.21 The prospect of a safe and effective therapeutic vaccine could be transformative; however, cancer22 vaccines have had limited success, in part due to the lack of CD8+ T-cell response elicited by vaccination23 (7). Many vaccine modalities have had success in the generation of antibody and CD4 responses (8, 9,24 10), however, despite some promising preclinical data (11, 12), there has been limited CD8 infiltration25 and subsequent clinical benefit. A significant consideration is that most vaccine strategies primarily rely26Journal Pre-proof on cross-presentation of antigen that is endocytosed by a resident dendritic cell for cytotoxic or effector T1 cell activation. Endocytosis is the process by which cells engulf material, resulting in the material being2 segregated from the cytosol in an endosome. This results primarily in MHC Class II presentation and3 subsequent CD4+ helper T cell activation. Through a process referred to as cross-presentation, a small4 amount of endosomal material can be presented on MHC Class I to yield some CD8+ T cell activation5 (13). However, this process is inefficient, resulting in suboptimal priming of CD8+ T cells responses with6 anti-tumor or anti-viral activity.7 Therefore, to achieve effective CD8 responses one must likely provide significant antigen load to the8 patient with these technologies. While this has been possible in murine systems, scaling to humans has9 been infeasible (Table 2). In some cases, the human dose is the same, or very similar to a murine dose on10 a total mass basis – therefore dramatically lower on a surface area or mass scaling basis (11, 12, 14).11 By solving some of the underlying problems related to the enablement of MHC-I presentation to CD8 T12 cells, Cell Squeeze® technology may provide a path to achieving a more potent therapeutic cancer13 vaccine. In preclinical models, Cell Squeeze® has demonstrated the ability to induce robust MHC-I14 presentation across multiple target antigens, subsequently creating strong CD8+ T cell responses capable15 of tumor infiltration (15). This review explores the potential impact of this approach.16 Cell Squeeze® Technology17 Using the Cell Squeeze® microfluidics technology, cargo can be delivered intracellularly. Cells are18 flowed in a solution of cargo at high speeds through a microfluidic constriction that is smaller than the19 diameter of the cell which results in the temporary disruption of the cell membrane (Figure 1) (16). This20 enables the target cargo to enter directly into the cytosol of the cells. Cell Squeeze® can be used to21 deliver many types of molecules, including peptides, proteins, small molecules, nucleic acids, and gene-22 editing complexes, to a diversity of cell types (17). Many of these material classes, such as proteins,23 peptides, and nucleic acids, have been difficult to deliver with existing delivery techniques. Additionally,24 multiple materials can be delivered into the cells simultaneously enabling the ability to multiplex and25 engineer several cellular functions in a single step.26Journal Pre-proof In the context of dendritic cells, for example, direct cytosolic delivery of protein antigen (Ag) was shown2 to be ∼1000 times more effective than cross-presentation after endocytic uptake of protein Ag (15). These3 engineered cells also showed potent abilities to stimulate previously activated CD8+ T cells, both in vitro4 and in vivo (Figure 2). We also demonstrate that microfluidic squeezing can enable antigen presentation5 by human cells at a manufacturing scale for potential clinical application. Finally, we show that immune6 responses elicited by squeezed cells, in combination with an adjuvant, are capable of driving antitumor7 effects that correlate with an influx of tumor-specific CD8+ T cells. By overcoming the fundamental8 barrier to effective MHC-I presentation of Ag, squeeze-engineered cells could potentially be used as the9 basis for a potent, rapid turnaround, cell-based vaccine that is applicable across tumor types (15).10 When assessed in vivo, mouse splenocytes squeezed with less than 100 ng of antigen elicit an anti-tumor11 response that is better than subcutaneous injection of 150 μg of the same antigen – a difference of three12 orders of magnitude(15). The first clinical candidate SQZ-PBMC-HPV is in a Ph 1/2 study in patients13 with HPV16+ recurrent, locally advanced, or metastatic solid tumors as a monotherapy and in14 combination with ICIs. The preliminary findings are discussed later in this review. The product is based15 upon ex vivo engineering of autologous peripheral blood mononuclear cells (PBMCs) to create antigen-16 presenting cells (APCs) which are then transferred back to the patient.17 A significant challenge for existing cell therapies is the time, cost, and complexity of manufacturing19 (Table 3). By potentially enabling rapid, cost-effective manufacturing of cell therapies, the Cell20 Squeeze® technology could potentially improve the feasibility and accessibility of cell therapies for many21 indications. The current SQZ process for producing clinical material is a fully closed system that22 maintains aseptic process conditions. The system utilizes a disposable kit that includes the microfluidic23 chips and incorporates integrity tests on the kit prior to the initiation of the manufacturing process. The24 microfluidic chip has hundreds of parallel constriction channels. The manufacturing time for the current25 sterile product candidate is under 24 hours, with a vein-to-vein time of approximately one week.26Journal Pre-proof Sipuleucel-T (Provenge®) has a shorter vein-to-vein time (~3 days), however drug product is released at1 risk; final sterility information is not available until after the patient has received drug, at least 7 days2 after the start of the manufacturing process (18). Current sterile cell therapies typically can have vein-to-3 vein times of four to six weeks.4 The automation of the entire manufacturing process is underway to create a point-of-care system to5 further reduce the manufacturing time and cost. By integrating upstream and downstream manufacturing6 operations with the Cell Squeeze® system, a fully closed, automated system suitable for on-site cell7 therapy production without a cleanroom is potentially enabled. This decentralized manufacturing8 approach is designed to enable rapid access to a cell-therapy intervention at community sites or field9 clinics, reduce treatment time from days to hours and improve patient access.10 Currently, Cell Squeeze® technology has been used to create drug products and is being tested in two11 ongoing clinical trials with the APC platform (NCT 04084951) and with the Activating Antigen Carrier12 (AAC) platform (NCT 04892043).13 The development plan of a therapeutic vaccine using the Cell Squeeze® technology aims to demonstrate14 proof-of-concept as monotherapy and also enhanced efficacy in combination with immune checkpoint15 inhibitors that are presently part of the therapeutic arsenal for HPV-driven tumors.16 ICIs, in particular those acting on the PD-1/PD-L1 axis, have been effective in treating multiple tumors,17 including HPV-positive tumors, by allowing the exhausted T-cells to once again recognize the malignant18 cells. However, only a subset of patients treated with ICIs responds and most eventually experience19 disease progression. The combination of a therapeutic vaccine with anti-PD-1 checkpoint inhibitors has20 been evaluated in HPV-induced cancers with promising results (19). Already, combination treatment21 with ICI and interleukin-2 (IL-2) has demonstrated preliminary efficacy in patients refractory to ICI22 monotherapy (20), suggesting that patients may be able to derive additional benefit by enhancing the23 tumor-specific T-cell response in combination with ICI treatment. The vaccine approach is expected to24 induce antigen-specific anti-tumor immune response that would work synergistically with the checkpoint25 inhibitors in removing the brakes of the CD8+ T cells and restoring the ability to attack the tumor. In26Journal Pre-proof preclinical studies, recombinant anti-PD-1 IL-2v fusion protein (PD1-IL2v) and M-SQZ-PBMC-HPV1 resulted in dramatic infiltration of E7-specific CD8+ T-cells into the TME and subsequent cures in treated2 animals (Figure 3-4) (21). The mechanism of action is thought to be targeted delivery of IL-2 to PD-13 expressing cells and these results highlight the potential of multiple immune modulating modalities, in4 this case, a cytokine and checkpoint inhibitor, to behave synergistically with an effective vaccine.5 The current clinical APC platform drug product, SQZ-PBMC-HPV, has completed the monotherapy7 dose-escalation portion of the study. SQZ-PBMC-HPV monotherapy was found to be safe and well-8 tolerated; no dose-limiting toxicities were observed (22). Encouraging anti-tumor activity was observed in9 patients treated with SQZ-PBMC-HPV monotherapy. Currently, the study is enrolling into the10 combination phase of the study, in which participants will be treated with SQZ-PBMC-HPV and an ICI.11 While SQZ-PBMC-HPV has shown promising results in the clinic, one can leverage the flexibility of the12 Cell Squeeze® technology to engineer additional stimulatory domains in cell therapy. SQZ-eAPC-HPV, a13 next-generation product, improves upon the first by engineering costimulatory and cytokine expression14 simultaneously with antigen presentation (Figure 5). This is accomplished by squeezing mRNA15 transcripts for E6, E7, CD86, membrane-bound IL-2, and membrane-bound IL-12 into PBMCs – resulting16 in a multi-functional cell therapy capable of more powerful T cell modulation.17 If successful in these initial studies, the Cell Squeeze® cancer vaccine strategies could be rapidly adapted19 for implementation across applications beyond HPV-driven cancers. The technology’s flexibility around20 antigen cargo enables the creation of therapeutic vaccines for immuno-oncologic targets such as Epstein-21 Barr virus (EBV), mutant KRAS, mutant TP53, and patient-specific neoantigens (7). Moreover, the22 clinical safety results to-date are supportive of the potential implementation of these therapeutic concepts23 in early-line settings, including combination approaches with currently approved ICIs. This provides the24 opportunity to complement or displace standard of care regimens with cancer vaccines that have the25 potential for durable patient impact.26Journal Pre-proof The ability to more directly engineer MHC-I presentation marks a potentially transformative advancement1 for the cancer vaccine field. As ongoing studies continue to explore this potential, careful consideration of2 biomarkers and trial design factors can help improve our understanding of the impact of this technological3 advance and how best to harness it for improved patient outcomes. Continued investment in future4 generations of multifunctional cell therapies, such as the eAPCs, can further enhance the ultimate5 probabilities of success while the development of more streamlined manufacturing can broaden the6 accessibility of these cell therapies. Ultimately, this latest generation of Cell Squeeze®-enabled cancer7 vaccines may finally achieve the patient impact this field has long aspired to.8 Author contributions10 All authors contributed to the article and approved the submitted version11 Funding information13 This work was not supported by any funding14 References17

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### Highlights

 Cell Squeeze® is a novel technology that relies on temporarily disrupting the cell membrane to deliver cargo directly into the cytosol  By enabling direct cytosolic delivery, Cell Squeeze® enhances MHC Class I presentation of antigens for CD8+ T cell activation  With rapid, cost-effective manufacturing of cell therapies, the Cell Squeeze® technology could potentially improve the feasibility and accessibility of cell therapies for many indications.Journal Pre-proof

1. Cells and target

cargo together in suspension

1. Cells are squeezed

through SQZ chip at high speeds

1. Cell membranes

are temporarily disrupted

1. Target cargo

enters the cytosol of the cells

1. Membranes

resealPBMCs RBCs HSC / iPSCs Applicable to diverse cell types Proteins and peptides Gene editing complexes Nanomaterials Nucleic acids Small Molecules Robust across material classes Electroporation (EP) results in substantial gene misregulation vs Squeezing

### Ep Sqz

Preservation of cell function A. B.B.B. C.C. D.Journal Pre-proof Antigen endocytosis CD4 Helper T Cell antibody responses

### Mhc-ii

### Mhc-i

B. Traditional cancer vaccines rely on cross-presentation Squeezed antigen Primarily

### Mhc-i

CD8 Killer T Cell activation A. SQZ® APC Mechanism enables direct antigen presentation C. Cell Squeeze® is approximately 1000x more potent than cross presentation at stimulating T-cell activation in dendritic cellsJournal Pre-proof Figure 1. Cell Squeeze® Technology Overview There are two categories of approaches one can pursue with the Cell Squeeze® technology to engineer an anti-cancer vaccine:

1. Leveraging the direct cytosolic delivery capabilities of the technology to by-pass crosspresentation and facilitate direct MHC-I expression of target antigens
2. Using the ability to deliver material to red blood cells to transport material to the professional

DCs that are most adept at antigen presentation in vivo. This technology is currently in clinic, however since the mechanism of action is significantly different, it is not covered in this paper. A. Cell Squeeze® technology passes cells through a restriction point, temporarily disrupting the cell membrane, allowing for delivery of cargo. B. Cell Squeeze® technology is applicable to a broad array of cell types. Currently, RBC, PBMCs, and HSC/iPSCs are currently being evaluated as clinical candidates. C. A variety of cargos may be introduced into the cell, including proteins, peptides, nanomaterials, nucleic acids, and small molecules. D. Heat map showing gene misregulation in human T cells 6 hours after delivery of fluorescently labeled dextran via either electroporation or Cell Squeeze® (16).Journal Pre-proof Figure 2. Traditional Cancer Vaccine Endogenous Cross Presentation on MHC Class II vs Cell Squeeze® Cancer Vaccine Direct Presentation on MHC Class I A. Cell Squeeze® delivers cargo directly into the cytosol of the cell, resulting in primarily MHC Class I presentation. B. Traditional cancer vaccines have relied on endocytosis to process antigen, which results primarily in MHC Class II presentation. C. Control murine bone marrow-derived dendritic cells (BMDCs) and BMDCs Cell Squeezed® with fluorescently labeled OVA were cultured with CD8+ T cells for 24 hours, then assessed by flow cytometry (15). Cell Squeeze® is approximately 1000x more potent than cross-presentation at stimulating T-cell activation (23).Journal Pre-proof Figure 3. CD8+ Infiltration into the TME SQZ and Combo mice were treated with 1 x106 M-SQZ- PBMC-HPV 14 days after TC-1 inoculation. PD1-IL2v and Combo mice were dosed with 0.5mg/kg PD1- IL2v on day 21. Tumors were harvested on day 24 and TILs analyzed by flow cytometry (21).Journal Pre-proof Figure 4. M-SQZ-PBMC-HPV Vaccination has Synergistic Anti-Tumor Activity when Combined with ICI: Mice were implanted with TC-1 cells. For mice treated with M-SQZ-PBMC-HPV, vaccination occurred 14 days later. Mice receiving PD1-ILv2 were administered drug intravenously 21, 28, and 35 days after implantation. Greater tumor reduction was observed in mice treated with M-SQZ-PBMC-HPV and PD1-IL2v than in mice treated with M-SQZ-PBMC-HPV alone or PD1-IL2v,suggesting there is a synergistic drug effect (21).Journal Pre-proof Figure 5: Anticipated Clinical Translation of Multi-functional eAPCs. Since SQZ-eAPC-HPV is manufactured by using Cell Squeeze® to insert mRNA that will translate full-length E6, full-length E7, membrane bound CD86, membrane bound interleukin-2, and membrane bound interleukin-12, SQZeAPC-HPV is expected to have enhanced CD8+ activation and broader antigen repertoire than SQZ- PBMC-HPV, which is manufactured using only the immunodominant synthetic long peptides for E6 and E7. SQZ-eAPC-HPV is also expected to have enhanced signal 1 (due to E6 and E7), signal 2 (due to CD86), and signal 3 (due to interleukin-2 and interleukin-12).Journal Pre-proof Table 1. Comparison of Different Approaches to Intracellular Delivery Table 1. Many intracellular delivery techniques have been developed, these are covered in detail in Stewart et al, however the most commonly used approaches in current cell therapy manufacturing are viral transduction or electroporation (24). Viral Electroporation Cell Squeeze® Minimal Cell perturbation Tendency to trigger innate anti-viral responses. Risk of integration of viral components into the genome Disruption of normal gene expression that can interfere with important cell functions such as differentiation, expansion, cytokine secretion, etc. Minimal perturbation of gene expression or normal cell functions Scalability Production of GMP virus with sufficient batch-to-batch consistency can be a challenge Flow-based systems are scalable, cuvette-based systems have challenges Scalable through channel parallelization Universal across cell types Only applicable to certain cell types and often require different viral constructs for different cell types Only applicable to certain cell types Applicable to all mammalian cell types attempted to date Material independent No, can only deliver nucleic acids compatible with viral packaging No, complex delivery mechanism favors highly charged materials such as nucleic acids Diffusion mediated delivery is mostly cargo agnostic. Relatively large (>100nm) nanoparticles can become a challenge Compatible with intracellular targeting No, viral delivery mechanism provides little flexibility Some flexibility to accommodate various targeting domains to direct cargo to different intracellular locations Yes Dosage Control Stochasticity of viral transduction can lead to high variability Some control, however non-linear optimization space of EP parameters poses a challenge Predictable diffusion-mediated mechanism ensures a fairly linear control by adjusting material concentration in the buffer Cost High: production of viral material is complex and expensive Low LowJournal Pre-proof Table 2. Comparison Preclinical vs Clinical Dosing in Different HPV16 Cancer Vaccines SQZ APCs (SQZeAPC-HPV) BioNTech mRNA (HPV16

### Rna-lpx)

### Isa (isa101)

Mouse Dose1 40x106 cells/kg (18) 1,600ug/kg (11) 6,000ug/kg (12) Human High Dose2 10x106 cells/kg3 1ug/kg (25, 26) 4.3ug/kg (14) Fold Difference 4x 1,600x 1,400x 1Assumes a 25g mouse. 2Assumes a 70kg human. 3Intended high dose, not yet tested in clinic. Table 2. A comparison of preclinical and clinical doses in different HPV16 cancer vaccines. The preclinical doses of BioNTech and ISA vaccines were 1,000x greater than the high doses used in human trials; scaling preclinical doses to humans has been infeasible.Journal Pre-proof Table 3. Overview of Cancer Vaccine Manufacturing Cell Type Drug Product Cell Yield Manufacturing time Estimated veinto-vein Drug Product Release Characterization Other Parameters DCs Sipuleucel-T (18) 50-100M cells 3-4 days ~3 days1 At risk 3 leukaphereses PBMCs SQZ-PBMC-HPV 5-10B cells <24hrs ~1 week Sterile 1 leukapheresis RBCs SQZ-AAC-HPV TBD <24hrs ~1 week Sterile 1 whole blood collection CAR-T Tisagenlecleucel (27, 28) 60-600M ~23 days ~3-8 weeks Sterile 1 leukapheresis TIL TIL (29) >1B ~20 days ~30 days At risk Isolated from resected metastatic lesion 1 Sipuleucel-T has an estimated vein-to-vein time of ~3 days. This rapid vein-to-vein time is due to the product being released before microbial and sterility testing is resulted i.e., at risk. Vein-to-vein time would be at least 7 days (sterility test incubation time) otherwise. Table 3. A summary of cancer vaccines using different cell types. TIL and CAR-T vaccines have a lengthy manufacturing due to the ex vivo expansion of cells, leading to longer vein-to-vein times for patients and increased cost. TIL and DC vaccines are released before all quality control testing is complete i.e., at risk. TIL and DC vaccines also have more involved cell harvesting processes than other vaccines. Cancer vaccines developed using Cell Squeeze technology are manufactured using only one blood collection (either whole blood or leukapheresis), have a rapid manufacturing time, and are confirmed sterile at the time of release.Journal Pre-proof

## Provenance

Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.