The development of antibody-based immunotherapies has, compared with conventional therapeutic strategies patients, transformed the therapeutic landscape in melanoma, non-small cell lung cancer, bladder cancer, and other cancers. Many of these novel agents have shown potent antitumor activity, resulting in deep and durable antitumor effects.

For example, while the median survival of patients with metastatic melanoma was historically <1 year and long-term survivorship rarely seen, [1] with the advent of immune checkpoint inhibitors and effective targeted therapies, survival rates for patients with metastatic melanoma have dramatically increased and durable disease control is a real possibility.[2]

Combination immunotherapy with the PD-1 blocking antibody, nivolumab (Opdivo®; Bristol Meyers Squibb), and anti-CTLA-4, ipilimumab (Yervoy®; Bristol Meyers Squibb), has demonstrated higher response rates than either therapy alone in metastatic melanoma. However, dual checkpoint blockade causes more frequent and severe immune-related adverse events (irAEs) [3] And while the therapeutic results of immune checkpoint inhibitors are compelling, these relatively new agents have been associated with severe immune-related adverse events, including rash, diarrhea, colitis, hypophysitis, hepatotoxicity, and hypothyroidism, which, in some cases may lead to high morbidity, are potentially life-threatening, and limit the duration of treatment.

Research has further shown that the incidence of severe immune-related adverse events increases when programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) inhibitors are combined with anti-CTLA-4 and/or other multidrug regimens resulting in on-target, off-tumor toxicity of anti-CTLA4 checkpoint inhibitors leads to severe adverse events, restricting therapeutic efficacy. For example, the additive toxicity observed with the clinically validated PD1 and CTLA4 combination therapy can be life-threatening to many patients even at a fraction of their doses when given as monotherapy.[4][5]

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Reducing on-target, off-tumor toxicity
To reduce the on-target, off-tumor toxicity of anti-CTLA4 checkpoint inhibitors, scientists at BioAtla have engineered anti-CTLA4 antibodies and generated a new class of antibodies called conditionally active biologic (CAB) antibodies.

This new class of antibodies uses physiological chemicals (bicarbonate, hydrogen sulfide) as protein-associated chemical switches (PaCS) to reduce binding under normal physiological conditions while maintaining binding in the tumor. In essence, the novel technology makes these antibodies active only in the acidic tumor microenvironment while the binding is reversibly inhibited in healthy tissue. This improved tumor targeting utilizes a newly discovered chemical switch system and is shown in animal models to provide for potent anti-tumor activity with markedly reduced toxicity to normal tissue, indicating a widened therapeutic index.

In a recent edition of Proceedings of the National Academy of Sciences (PNAS) the scientists describe the design and functionality of these novel therapeutic antibody candidates using BioAtla’s proprietary CAB technology. [6]

Scientists at Bioatla have applied the CAB technology in the preclinical development of the company’s immuno-oncology based CAB-CTLA4 antibody candidates as well as several CAB antibodies directed against other important oncology targets.

“CAB antibodies utilize a newly discovered switch mechanism that allows them to be active only in the tumor microenvironment and not active under normal physiological conditions. CAB antibodies demonstrate reduced peripheral toxicity and therefore are expected to provide a wider therapeutic window compared to traditional antibodies currently available for cancer therapy, potentially enabling higher dosing and longer treatments for improved efficacy,” explained co-inventor Jay M. Short, Ph.D., Chairman, Chief Executive Officer and co-founder of BioAtla, Inc.

Warburg Effect
The CAB technology capitalizes on the well-established Warburg Effect* that through a glycolytic process leads to an acidic external tumor microenvironment. [7] Extracellular pH levels in tumors have been measured to be as low as pH5.8 compared to the tightly controlled, alkaline, pH7.4 of blood, with even higher pH in healthy tissues. Glycolytic metabolism is also the basis of the established PET scanning technology for cancer detection for tumor types. CAB proteins have increased binding activity as the pH in the microenvironment becomes acidic, while being inactive in normal physiological environments.

Scientists at BioAtla discovered a novel chemical switch mechanism involving physiological-occurring chemicals, such as bicarbonate and hydrogen sulfide. These molecules are negatively charged at physiological conditions and interact with positively charged areas on the protein surface. Under acidic conditions of the tumor microenvironment, they are neutralized and released from the protein surface, uniquely allowing CAB antibodies to bind to their target and attack the tumor cell. The scientists refer to this novel physiological mechanism, used for generating CABs, as Protein-associated Chemical Switch(es) or PaCS mechanism.

Tumor-selective therapeutics
CAB antibodies belong to a novel class of tumor-selective therapeutics that do not require the addition of a protective group and irreversible enzymatic activation in the tumor that is used with prodrug designs. The CAB-CTLA4 candidates described in the paper showed substantially reduced binding at pH7.4 compared to binding at pH6.0, while the comparable Ipilimumab analog (IpA) binding showed no dependence on pH, thereby leading IpA to bind and attack normal cells, which results in dangerous on-target off-tumor toxicity. In comparison, multiple CAB candidates demonstrated substantial binding differentials between pH6.0 and pH7.4 conditions ranging from 9-fold to over 175-fold by ELISA, which is expected to lead to an improved therapeutic index and the potential improved clinical risk-benefit in future therapies.

The ability to design CAB tumor target binding for a specific range of pH conditions demonstrates the flexibility provided by the PaCS mechanism and the CAB technologies. The selection of a CAB antibody candidate is based upon strong differential pH binding between tumor and normal cells that can lead to increased anti-tumor potency with reduced toxicity, while maintaining a low immunogenicity risk and efficient manufacturing characteristics.

In addition to the development of CAB-CTLA4 discussed in the paper, BioAtla has successfully generated several CAB antibodies against multiple targets including EpCAM, Her2, Nectin-4, and CD73. The proprietary technology has also successfully been used for the development of antibody-drug conjugates (ADCs) and T-cell engaging bispecific antibodies. The ability to design conditionally active therapeutics with stronger selectivity over narrower pH ranges using the PaCS mechanism offers the opportunity to greatly enhance both the safety and potency of future therapies for solid tumors.

Potential for additional therapeutic modalities
It is expected from the studies described in the paper that there is a potential for other yet-to-be-identified PaCS molecules in disease-related microenvironments, whether controlled through pH, concentration, or other molecular characteristics (intra- or intermolecularly) for enhancing a drug’s therapeutic index. Potential new therapeutic candidates addressing these opportunities are not limited to antibodies, but also include small molecules, encompassing lipids, sugars, and nucleic acid-based agents or drugs.

Based on their ongoing work, the scientists expect that PaCS protein-chemical systems are important naturally occurring regulatory systems linked to a range of disease-related microenvironments, including cancer, inflammation, and cellular senescence.

Development program
BioAtla has two CAB programs currently in Phase II clinical testing in the United States, BA3011, a novel conditionally active AXL-targeted antibody-drug conjugate (CAB-AXL-ADC), and BA3021, a novel conditionally active ROR2-targeted antibody-drug conjugate (CAB-ROR2-ADC). BioAtla’s investigational CAB CTLA-4 antibody, BA3071, is the subject of a global co-development and collaboration agreement with BeiGene Ltd. for its development, manufacturing, and commercialization. BA3071 is a novel, CTLA-4 inhibitor that is designed to be conditionally activated in the tumor microenvironment in order to reduce systemic toxicity and potentially enable safer combinations with checkpoint inhibitors such as anti-PD-1 antibodies.

Note
* The Warburg Effect is defined as an increase in the rate of glucose uptake and preferential production of lactate, even in the presence of oxygen. Cancer cells rewire their metabolism to promote growth, survival, proliferation, and long-term maintenance. The common feature of this altered metabolism is the increased glucose uptake and fermentation of glucose to lactate. This phenomenon is observed even in the presence of completely functioning mitochondria and, together, this is known as the Warburg Effect.[7]

Clinical trials
CAB-ROR2-ADC Safety and Efficacy Study in Patients With Solid Tumors – NCT03504488
CAB-AXL-ADC Safety and Efficacy Study in Adult With NSCLC – NCT04681131
CAB-AXL-ADC Safety and Efficacy Study in Adult and Adolescent Patients With Solid Tumors – NCT03425279

Reference
[1] Korn EL, Liu PY, Lee SJ, Chapman JA, Niedzwiecki D, Suman VJ, Moon J, Sondak VK, Atkins MB, Eisenhauer EA, Parulekar W, Markovic SN, Saxman S, Kirkwood JM. Meta-analysis of phase II cooperative group trials in metastatic stage IV melanoma to determine progression-free and overall survival benchmarks for future phase II trials. J Clin Oncol. 2008 Feb 1;26(4):527-34. doi: 10.1200/JCO.2007.12.7837. PMID: 18235113.
[2] Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, Sharfman WH, Brahmer JR, Lawrence DP, Atkins MB, Powderly JD, Leming PD, Lipson EJ, Puzanov I, Smith DC, Taube JM, Wigginton JM, Kollia GD, Gupta A, Pardoll DM, Sosman JA, Hodi FS. Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab. J Clin Oncol. 2014 Apr 1;32(10):1020-30. doi: 10.1200/JCO.2013.53.0105. Epub 2014 Mar 3. PMID: 24590637; PMCID: PMC4811023.
[3] Pollack MH, Betof A, Dearden H, Rapazzo K, Valentine I, Brohl AS, Ancell KK, Long GV, Menzies AM, Eroglu Z, Johnson DB, Shoushtari AN. Safety of resuming anti-PD-1 in patients with immune-related adverse events (irAEs) during combined anti-CTLA-4 and anti-PD1 in metastatic melanoma. Ann Oncol. 2018 Jan 1;29(1):250-255. doi: 10.1093/annonc/mdx642. PMID: 29045547; PMCID: PMC5834131.
[4] Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, Schadendorf D, Dummer R, Smylie M, Rutkowski P, Ferrucci PF, Hill A, Wagstaff J, Carlino MS, Haanen JB, Maio M, Marquez-Rodas I, McArthur GA, Ascierto PA, Long GV, Callahan MK, Postow MA, Grossmann K, Sznol M, Dreno B, Bastholt L, Yang A, Rollin LM, Horak C, Hodi FS, Wolchok JD. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015 Jul 2;373(1):23-34. doi: 10.1056/NEJMoa1504030. Epub 2015 May 31. Erratum in: N Engl J Med. 2018 Nov 29;379(22):2185. PMID: 26027431; PMCID: PMC5698905.
[5] Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Rutkowski P, Lao CD, Cowey CL, Schadendorf D, Wagstaff J, Dummer R, Ferrucci PF, Smylie M, Hogg D, Hill A, Márquez-Rodas I, Haanen J, Guidoboni M, Maio M, Schöffski P, Carlino MS, Lebbé C, McArthur G, Ascierto PA, Daniels GA, Long GV, Bastholt L, Rizzo JI, Balogh A, Moshyk A, Hodi FS, Wolchok JD. Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N Engl J Med. 2019 Oct 17;381(16):1535-1546. doi: 10.1056/NEJMoa1910836. Epub 2019 Sep 28. PMID: 31562797.
[6] Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Hwai Wen Chang, Gerhard Frey, Haizhen Liu, Charles Xing, Lawrence Steinman, William J. Boyle, Jay M. Short. Proceedings of the National Academy of Sciences Mar 2021, 118 (9) e2020606118; DOI: 10.1073/pnas.2020606118
[7] Liberti MV, Locasale JW. The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci. 2016 Mar;41(3):211-218. doi: 10.1016/j.tibs.2015.12.001. Epub 2016 Jan 5. Erratum in: Trends Biochem Sci. 2016 Mar;41(3):287. Erratum in: Trends Biochem Sci. 2016 Mar;41(3):287. PMID: 26778478; PMCID: PMC4783224.

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