Pravin T. P. Kaumaya

Supplemental Figure 1: Anti-tumor mechanisms investigations

Cancer immunotherapy with checkpoint inhibitors has resulted in impressive clinical results in several cancer indications. Despite this success, only a fraction of patients show durable and complete response to blockade by ipilimumab, an anti–cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) mAb. We identified four CTLA-4 peptide sequences from which engineered chimeric B-cell epitope constructs incorporating a “promiscuous” T-cell epitope elicited highly immunogenic anti–CTLA-4 natural polyclonal antibodies by immunization. Combination of CTLA-4 peptide vaccine with other checkpoint inhibitor vaccines PD1-Vaxx or PDL1-Vaxx was investigated in several breast and colon carcinoma BALB/c syngeneic models (CT26, 4T1, and D2F2). CTLA-4 vaccines showed significant tumor suppression and prolonged survival rates as compared with anti-mouse CTLA-4 mAb 9H10. The resulting antipeptide antibodies suppressed tumor proliferation and migration similar to ipilimumab. We focused on one CTLA-4 epitope sequence 130 to 150 that embodies the “MYPPPY” motif that ipilimumab binds to. Combination of MVF-CTLA-4 (130–150) with either PD1-Vaxx or PDL1-Vaxx showed synergistic activity. The 130 to 150 peptide mimic demonstrated that the polyproline type II helix motif showed inhibition of tumor growth and therapeutic efficacy in the syngeneic CT26/BALB/c model. Several CTLA-4 vaccines have been identified that show synergistic activities with other checkpoint inhibitor vaccines to PD1 and PDL1.

Immuno-oncology has revolutionized cancer treatment by mobilizing the immune system to eliminate tumors. Although immune checkpoint inhibitors and T cell therapies have mediated durable responses in hematologic malignancies and select solid tumors, most patients still relapse or fail to respond. To overcome these limitations, novel, next-generation immune constructs, including bispecific-cell therapy combinations, armored cells, tethered cytokines, immune-stimulatory antibody conjugates, and in vivo gene editing, are being developed to enhance specificity, persistence, and immune activation. This Society for Immunotherapy of Cancer roadmap highlights emerging technologies that integrate, redirect, or potentiate immune response. We examine advances in construct design, strategies for clinical translation, and opportunities for combinatorial approaches. By addressing translational barriers and real-world challenges, we outline how innovative engineering can unlock a new era of safe, durable, and accessible immunotherapies. Realizing this potential will require coordinated efforts from researchers, clinicians, industry leaders, and policymakers to deliver curative outcomes to patients worldwide.

Cancer immunotherapy with checkpoint inhibitors has resulted in impressive clinical results in several cancer indications. Despite this success, only a fraction of patients show durable and complete response to blockade by ipilimumab, an anti–cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) mAb. We identified four CTLA-4 peptide sequences from which engineered chimeric B-cell epitope constructs incorporating a “promiscuous” T-cell epitope elicited highly immunogenic anti–CTLA-4 natural polyclonal antibodies by immunization. Combination of CTLA-4 peptide vaccine with other checkpoint inhibitor vaccines PD1-Vaxx or PDL1-Vaxx was investigated in several breast and colon carcinoma BALB/c syngeneic models (CT26, 4T1, and D2F2). CTLA-4 vaccines showed significant tumor suppression and prolonged survival rates as compared with anti-mouse CTLA-4 mAb 9H10. The resulting antipeptide antibodies suppressed tumor proliferation and migration similar to ipilimumab. We focused on one CTLA-4 epitope sequence 130 to 150 that embodies the “MYPPPY” motif that ipilimumab binds to. Combination of MVF-CTLA-4 (130–150) with either PD1-Vaxx or PDL1-Vaxx showed synergistic activity. The 130 to 150 peptide mimic demonstrated that the polyproline type II helix motif showed inhibition of tumor growth and therapeutic efficacy in the syngeneic CT26/BALB/c model. Several CTLA-4 vaccines have been identified that show synergistic activities with other checkpoint inhibitor vaccines to PD1 and PDL1.

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer patients and has become a mainstay approach in modern cancer immunotherapy. However, only a subset of patients respond to checkpoint monoclonal antibody immunotherapy. Therefore, it is urgent to develop new therapeutic strategies against cancer. A novel B-cell peptide epitope PDL1 (programmed death ligand 1) cancer vaccine has been developed, with amino acids 130-147 linked to the MVF peptide ("promiscuous" T-cell measles virus fusion protein) via a GPSL linker. Preclinical testing has indicated that this PDL1 vaccine (PDL1-Vaxx) effectively stimulates highly immunogenic antibodies in animals. Animals immunized with PDL1-Vaxx show reduced tumor burden and extended survival rates in various animal cancer models. The mechanisms of action indicate that vaccine-elicited antibodies inhibit tumor cell proliferation, induce apoptosis, and block the PD-1/PD-L1 interaction. This manuscript introduces a magnetic bead-based assay that uses a dual-reporter flow analysis system to evaluate the PD-1/PD-L1 interaction and its blockade by the anti-PDL1 antibodies raised against the PDL1-Vaxx.

Blockade of checkpoint receptors with monoclonal antibodies against CTLA-4, PD-1 and PD-L1 has shown great clinical success in several cancer subtypes, yielding unprecedented responses albeit a significant number of patients develop resistance and remain refractory. Both PD-1/PD-L1 and HER-2 signaling pathway inhibitors have limited efficacy and exhibits significant toxicities that limit their use. Ongoing clinical studies support the need for rationale combination of immuno-oncology agents to make a significant impact in the lives of cancer patients. We introduce the development of a novel chimeric PD-L1 B-cell peptide epitope vaccine (amino acid 130–147) linked to a “promiscuous” T cell measles virus fusion (MVF) peptide (MVF-PD-L1(130); PDL1-Vaxx) or linked to tetanus toxoid (TT3) TT3-PD-L1 (130) via a linker (GPSL). These vaccine constructs are highly immunogenic and antigenic in several syngeneic animal models. The PD-L1 vaccines elicited high titers of polyclonal antibodies that inhibit tumor growth in multiple syngeneic cancer models, eliciting antibodies of different subtypes IgG1, IgG2a, IgG2b and IgG3, induced PD-1/PD-L1 blockade, decreased proliferation, induced apoptosis and caused ADCC of tumor cells. The PDL1-Vaxx induces similar inhibition of tumor growth versus the standard anti-mouse PD-L1 antibody in both syngeneic BALB/c and C57BL/6J mouse models. The combination of PDL1-Vaxx with HER-2 vaccine B-Vaxx demonstrated synergistic tumor inhibition in D2F2/E2 carcinoma cell line. The anti-PDL1-Vaxx block PD-1/PD-L1 interaction and significantly prolonged anti-tumor responses in multiple syngeneic tumor models. The combination of HER-2 vaccine (B-Vaxx) with either PDL1-Vaxx or PD1-Vaxx demonstrated synergistic tumor inhibition. PDL1-Vaxx is a promising novel safe checkpoint inhibitor vaccine.

Immunotherapy with monoclonal antibodies to checkpoint inhibitors against the PD-1/PD-L1 signaling pathway is a landmark achievement in cancer therapy. Some anti-PD-1 inhibitors such as nivolumab and pembrolizumab have shown clinical success, in a percentage of patients with prolonged survival rates. However, adverse effects accompany these benefits. In this case, strategies with lower toxicity and increased specificity are urgently required. Cancer vaccines have the ability to stimulate the native immune system and in particular, an engineered B-cell epitope can elicit high-affinity polyclonal antibodies with similar efficacy to PD-1 monoclonal antibodies in murine animal models. We have previously designed and synthesized a unique B-cell vaccine, PD1-Vaxx [MVF-PD-1(92-110)], and we have tested the immunogenicity and antitumor properties in CT26 colon cancer BALB/c syngeneic mice model. This manuscript provides results from comprehensive preclinical pharmacology studies encompassing primary and secondary pharmacodynamics, biodistribution, and safety studies. The results from these preclinical studies support the use of PD1-Vaxx in a first-in-human clinical trial in patients with non-small cell lung cancer (NSCLC). A phase I trial in patients with NSCLC has commenced.

We developed a PD-1 B-cell epitope vaccine (PD1-Vaxx) to rival nivolumab therapy which has received ethics approvals for a Phase 1 clinical trial in Australia. The US FDA granted Investigational New Drug approval to Imugene Ltd for clinical testing in NSCLC. We demonstrated synergistic vaccine combinations with an HER-2 targeted vaccine (B-Vaxx).

In light of the numerous US FDA-approved humanized monoclonal antibodies (mAbs) for cancer immunotherapy, it is surprising that the advancement of B-cell epitope vaccines designed to elicit a natur... Graphical abstract

Therapeutic blockade of PD-1/PD-L1 signaling with monoclonal antibodies (mAbs) has shown clinical success and activity across a broad set of cancer subtypes. However, monotherapy with PD-1/PD-L1 inhibitors are only effective in a subset of patients and ongoing studies show efficacy of treatment depends on a combinatorial approach. Contrary to mAbs chimeric B-cell cancer vaccines incorporating a “promiscuous” T-cell epitope have the advantage of producing a polyclonal B-cell antibody that can potentially induce memory B- and T-cell responses, while reducing immune evasion and suppression. Here, we describe a novel PD-1 B-cell peptide epitope vaccine (amino acid 92–110; PD1-Vaxx) linked to a measles virus fusion peptide (MVF) amino acid 288–302 via a four amino acid residue (GPSL) emulsified in Montanide ISA 720VG that aims to induce the production of polyclonal antibodies that block PD-1 signaling and thus trigger anticancer effects similar to nivolumab. In preclinical studies, the PD1-Vaxx outperformed the standard anti-mouse PD-1 antibody (mAb 29F.1A12) in a mouse model of human HER-2 expressing colon carcinoma. Furthermore, the combination of PD1-Vaxx with combo HER-2 peptide vaccine (B-Vaxx) showed enhanced inhibition of tumor growth in colon carcinoma BALB/c model challenged with CT26/HER-2 cells. The PD-1 or combined vaccines were safe with no evidence of toxicity or autoimmunity.