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Bispecific antibodies which bind a tumor antigen and the CD3 heterodimer of the T cell receptor to form a synthetic immunological synapse represent a class of T cell engagers that has been clinically validated with the approvals of blinatumomab (Blinctyo®) and mosunetuzumab (Lunsumio®), targeting CD19 and CD20 respectively, for the treatment of B cell acute lymphoblastic leukemia and follicular lymphoma. Despite being a potent class of therapeutics, not all patients respond to therapy. Recent studies into state of blinatumomab-treated T cells highlight exhaustion and the lack of co-stimulation as potential factors in resistance. In solid tumors, the immunosuppressive tumor microenvironment is also a major factor while cytokine release is a safety consideration for all bispecifics that bind CD3 with strong affinity. To address the challenges of CD3 bispecifics, we have developed the EVOLVE™ platform, a trispecific antibody that consists of tumor targeting and attenuated CD3 binding affinity coupled with a CD2 agonist. We show that CD2 costimulation is superior to other pathways in its ability to sustain T cell activation and expansion and cytokine production over repeated cycles of stimulation. By integrating a CD2 agonist with an attenuated CD3 affinity bispecific, we can restore cytolytic potential without concomitant increase in cytokine release as compared to a strong CD3 affinity bispecific. The EVOLVE platform is also capable of inducing tumor cell killing by in vitro exhausted T cells whereas the bispecific did not. In terms of safety, the EVOLVE platform does not induce peripheral T cell activation and cytokine release in PBMCs in the absence of target as compared to CD3 and CD28 agonist antibodies. The superiority of the EVOLVE platform compared to a bispecific was also demonstrated in vivo where we observed 8/8 complete responses in EVOLVE-treated animals whereas the strong CD3 affinity bispecific showed no difference compared to isotype control using a CORL105 lung cancer xenograft model. We believe these experiments recapitulate the continual clinical challenges of using strong CD3 affinity bispecifics while demonstrating the advantages of the EVOLVE platform. Finally, we demonstrate the modular nature of the EVOLVE platform across diverse tumor antigens including B7H4, PSMA, CD20, and a novel squamous tumor antigen ULBP2. Together our data highlight the broad applications of the EVOLVE platform to improve T cell-mediated anti-tumor immunity and suggest its potential as an emerging, first-in-category immunotherapeutic strategy to address unmet medical needs in oncology. Citation Format: Mohosin Sarkar, Eric M. Tam, Guixian Jin, Shu Shien Chin, Abudukadier Abulizi, Oksana A. Sergeeva, Zengzu Lai, Evelyn Teran, Hayden Karp, Danielle Klaskin, Nana Adjoa Pels, Xingyue An, Jennifer Ziegler, Changqing Yuan, Maria Hackettt, Sonali Dhindwal, Amber Fearnley, Julio Rodriguez, Louis Matis, Jay S. Fine, Jeremy S. Myers. EVOLVETM: A novel T cell engager platform with integrated CD2 costimulation engineered for the treatment of immune suppressive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2971.

CD3-bispecifics are antibody-based therapies that can simultaneously bind to a tumor cell surface antigen and T cells to establish a synapse between the tumor and T cell and activate T cell to induce specific killing of the tumor cell. CD3-bispecifics have demonstrated clinical success in B cell acute lymphoblastic leukemia and follicular lymphoma with approvals of that blinatumomab and mosunetuzumab that target B cell lineage antigens CD19 and CD20, respectively. However, clinical progress in deploying CD3-bispecifics for positive patient outcomes in solid tumors has been slow, due to tumor microenvironmental factors such as induction of T cell exhaustion, as well as the potential of CD3-bispecifics to mediate T cell anergy and dysfunction in the absence of adequate co-stimulation. Here we describe the development and preclinical validation of the EVOLVE platform, a tumor-targeted biologic that induces the formation of a synthetic synapse that simultaneously activates the T cell receptor complex and the CD2 receptor to optimize CD8 T cell effector phenotype and improve tumor cell killing ex vivo and in vivo, compared to matched CD3-bispecifics. We demonstrate that CD2 co-stimulation is superior to other forms of T cell co-stimulation in its ability to promote cytolytic co-stimulation, T cell cytokine production and T cell expansion. Furthermore, CD2 receptor expression is markedly elevated in tumor infiltrating lymphocytes across a broad set of tumor types, relative to the CD28 and 4-1BB costimulatory receptors. EVOLVE-mediated T cell activation is conditionally dependent on tumor antigen binding and can be tuned to promote optimal co-stimulation without increasing cytokine release relative to matched CD3-bispecifics. We also demonstrate the modular nature of the EVOLVE platform across diverse solid tumor antigens including B7H4 (VTCN1), and a novel squamous tumor antigen ULBP2. Our data highlight the broad applications of the EVOLVE platform to improve CD8 T cell-mediated anti-tumor immunity and suggest its potential as an emerging, first-in-category immunotherapeutic strategy to address unmet medical needs in oncology. Citation Format: Mandy Shu Shien Chin, Mohosin Sarkar, Eric Tam, Abudukadier Abulizi, Guixian Jin, Xingyue An, Evelyn Teran, Danielle M Klaskin, Nana Adjoa Pels, Maria Hackett, Oksana A Sergeeva, Hayden Karp, Julio Rodriguez, Sonali Dhindwal, Changqing Yuan, Zengzu Lai, Jennifer Zeiger, Amber Fearnley, Louis Matis, Jay Fine, Jeremy S Myers. EVOLVE: A novel costimulatory T cell engager platform engineered for the treatment of solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B44.

Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. TheHaemophilus influenzaetype b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM197conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM197conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.

Pseudomonas aeruginosa is a serious human pathogen in a variety of patient groups including those with burns, hospitalized in intensive care, cystic fibrosis and neutropenia. Since there is no vaccine available, passive antibody prophylaxis against protective epitopes is an alternative strategy to prevent P. aeruginosa infection. However, immunoglobulin derived from multiple donors has variable anti-pseudomonas antibody titers, and human Mab are difficult to make from patient samples. We previously reported the use of XenoMouse mice, Ig-inactivated transgenic mice reconstituted with human immunoglobulin loci, to generate human Mab against a single serotype of P. aeruginosa lipopolysaccharide O-specific side chain (PS). We now report the creation of a panel of anti-PS human IgG2 Mab against nine additional O-specific side chain P. aeruginosa serotypes. The majority of the Mab were highly opsonic for uptake and killing of homologous P. aeruginosa by human PMN in the presence of human complement, and all the Mab protected cyclophosphamide-induced neutropenic mice from fatal P. aeruginosa sepsis with homologous serotypes. DNA sequence analysis showed that the Mab used V(H)3, V(H)4, V(H)5 and V(H)6 and Vkappa2, 3 and 4 variable region genes consistent with the heterogeneity of P. aeruginosa LPS O-side chain structure. We conclude that human Mab made in these transgenic mice against common pathogenic serotypes of P. aeruginosa are opsonic and highly protective, and that a high titer, multi-valent human Mab preparation against the majority of circulating O-side chain serotypes of P. aeruginosa could be used as prophylaxis against invasive infections in selected patient groups.

The variable domain of heavy chain (VH) and light chain (VL) genes of anti-CD20 monoclonal antibody HI47 were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab’ fragment expression vector pYZFcd20. Chimeric anti-CD20 Fab’ fragment was expressed inE. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab’ fragment inhibited binding of HI47 to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab’ fragment inhibited the proliferation of Daudi cells, IC50 = 69 μg/mL. Affinity of chimeric anti-CD20 Fab’ fragment was determined, Ka was about 8.9×108 (mol/L)−1.

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

The production of a recombinant human IgG1 in transgenic tobacco was examined to determine whether a plant-derived antibody could recruit immune system effector function against a bacterial pathogen. A plant transformation vector was engineered to contain genes for a human kappa light chain and a human gamma-1 heavy chain with VHand VLsequences from a previously identified human IgG2 monoclonal antibody (MAb) that specifically binds to and opsonizesPseudomonas aeruginosaserotype O6ad. Unique NcoI and NotI restriction sites were incorporated to flank these variable sequences, resulting in a plant transformation vector that could be engineered for expression of any other human IgG1 antibody, requiring only the substitution of other VHand VLantigen-binding coding sequences. The plant-produced IgG1 was determined to have high-mannose glycan content and to be capable of mediating opsonophagocytosis ofP. aeruginosaserotype O6ad in vitro using human complement and human polymorphonuclear leukocytes. Thus, MAbs produced in plants from this vector could provide human IgG1 MAbs for targeting other pathogens that require the recruitment of immune system effector functions.

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI), induce no immunological memory and low Ab titers in infants. Conjugation of the Pn PS to the protein carrier CRM197 induces PS-specfic Ab in infants and memory similar to T dependent (Td) antigens. Glycoconjugates induce a Td PS immune response via antigen processing and presentation of carrier with MHC II, but the fate of the PS attached to the carrier is unclear. To determine the location of the PS of PnPS-CRM197 in the APC, we separately labeled PS and protein and tracked their location in APC. The PS of 19F-CRM197 was labeled by Alexa Fluor ® 594 hydrazide (labels PS red). The CRM197 was separately labeled in a reaction that did not label PS. Labeled 19F PS, 19F-CRM197 and CRM197 were incubated with 1x106 APC for 3–12 hours. APC were fixed and incubated with anti- HLA-DR Ab labeled by Alexa Fluor® 488 (green), followed by confocal microscopy. Labeled CRM197 was taken up and presented on the APC surface co-localized with MHC II (yellow; 42% positive cells). Labeled 19F PS was not internalized or presented on the surface. PS labeled 19F-CRM197 was internalized and co-localized on the surface with MHC II (23% positive). Brefeldin A and chloroquine blocked both CRM197 and 19F-CRM197 from co-localizing on the surface. These data suggest that PS of the 19F-CRM197 glycoconjugate enters the endosome, travels with CRM197 peptides to the APC surface and co-localizes with MHC II.