Trispecific binding proteins, methods, and uses thereof
Inventors
Beil, Christian • Beninga, Jochen • Birkenfeld, Joerg • Nabel, Gary J. • Qiu, Huawei • Rao, Ercole • Regula, Joerg • Seung, Edward • Wei, Ronnie • Wu, Lan • Xing, Zhen • Xu, Ling • Yang, Zhi-Yong • Cameron, Béatrice • Dabdoubi, Tarik • Lemoine, Cendrine • Prades, Catherine
Assignees
Sanofi SA • US Department of Health and Human Services
Publication Number
US-11613576-B2
Publication Date
2023-03-28
Expiration Date
2040-04-08
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Abstract
Provided herein are trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation, and wherein and a second pair of polypeptides possess a single variable domain forming a single antigen binding site. In some embodiments, the binding proteins comprise a binding site that binds a CD28 polypeptide, a binding site that binds a CD3 polypeptide, and a binding site that binds a third polypeptide, such as a tumor target protein. In some embodiments, the binding proteins comprise four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins. The disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins.
Core Innovation
Provided herein are trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites specifically binding one or more target proteins, with a first pair of polypeptides possessing dual variable domains having a cross-over orientation, and a second pair possessing a single variable domain forming one antigen binding site. The binding proteins can include binding sites for CD28, CD3, and a third polypeptide such as a tumor target protein (e.g., HER2, CD38) or HIV target proteins.
The binding proteins are designed to recruit T cells to tumor cells or virus-infected cells, subsequently activating the T cells via CD3 and CD28 binding sites to promote killing of adjacent tumor or infected cells, for example through a Granzyme/Perforin mechanism. This trispecific format offers a strategy to activate and expand antigen-specific effector and memory T cell populations, including central and effector memory cells, supporting treatment of cancer and chronic infectious diseases such as HSV, CMV, EBV, HIV-1, and HBV infections.
The background highlights the problem that monoclonal antibody therapies and bispecific antibodies have limitations in activating and expanding T cells effectively to treat cancer and chronic viral infections, including the need to eliminate viral reservoirs such as HIV-1 latently infected cells which survive current antiretroviral therapy. There is a need for novel therapeutic strategies to target and eradicate such reservoirs and tumors by simultaneously engaging multiple antigens to optimally activate T cells, including providing both antigen recognition and co-stimulation signals.
Claims Coverage
The claims disclose a binding protein comprising four polypeptide chains forming three antigen binding sites with specific structural and sequence features.
Binding protein comprising four polypeptide chains forming three antigen binding sites
A first polypeptide chain having the structure VL2-L1-VL1-L2-CL; a second polypeptide chain having the structure VH1-L3-VH2-L4-CH1-hinge-CH2-CH3; a third polypeptide chain having the structure VH3-CH1-hinge-CH2-CH3; and a fourth polypeptide chain having the structure VL3-CL, where the first and second polypeptides form a cross-over light chain-heavy chain pair.
First antigen binding site binds CD28 polypeptide
The first antigen binding site formed by VH1 and VL1 domains specifically binds a CD28 polypeptide, comprising defined CDR sequences or amino acid sequences as specified (e.g., SEQ IDs 49-54, 91-92).
Second antigen binding site binds CD3 polypeptide
The second antigen binding site formed by VH2 and VL2 domains specifically binds a CD3 polypeptide, comprising defined CDR sequences or amino acid sequences with variants of the CDR-L1 domain (e.g., SEQ IDs 55-57, 59-62, 93, 595, and VL sequence variants).
Third antigen binding site binds tumor target protein including CD38 polypeptide
The third antigen binding site formed by VH3 and VL3 domains specifically binds a tumor target protein, including a human CD38 polypeptide with defined CDR sequences or amino acid sequences (e.g., SEQ IDs 13-18, 19-24, 25-30, 31-36, 37-42, 43-48, and corresponding VH3 and VL3 sequences).
Inclusion of specific polypeptide sequences
Binding proteins comprise defined amino acid sequences for each polypeptide chain, with specific sequences or sequences having at least 95% identity, e.g., SEQ IDs 156-188 for the polypeptide chains composing the trispecific binding protein.
Linker sequences and lengths between domains
Linkers L1, L2, L3, and L4 are zero or selected from specified amino acid sequences (e.g., SEQ IDs 66-71); proportions and lengths vary with defined preferred lengths.
Fc region mutations in second and third polypeptide chains
Fc regions include human IgG4 or IgG1 domains with specific amino acid substitutions at positions corresponding to EU indexing, such as substitutions at positions 234, 235, 228, 409, 349, 354, 366, 368, and 407 to engineer knob-into-hole heterodimerization and reduce effector function (e.g., L234A, L235A, F234A, S228P, R409K, T366W, Y349C, L368A, Y407V).
The patent claims a trispecific binding protein with defined structural organization, antigen binding specificities (CD28, CD3, and tumor target such as CD38), specific variable domain sequences and linkers, and Fc region modifications to optimize stability, reduce unwanted effector function, and allow heterodimer formation.
Stated Advantages
Trispecific binding proteins provide specific targeting to tumor or HIV-infected cells while enabling optimal T cell activation by simultaneously providing antigen recognition and co-stimulation signals.
The trispecific format promotes expansion of antigen-specific CD4 and CD8 effector and memory T cell populations, including central and effector memory cells.
Anti-CD3 binding sites with high affinity human CD3 binding and reduced manufacturing liabilities are provided.
Binding proteins can bind both human and non-human primate CD38 polypeptides, enabling preclinical toxicological studies for safety evaluation.
Trispecific antibodies show effective in vitro cytotoxicity against multiple myeloma and lymphoma cells, with retained activity in presence of daratumumab.
Administration of Her2/CD28×CD3 trispecific antibody in tumor-bearing NSG mice leads to significant tumor regression without body weight loss and modulates tumor-infiltrating lymphocyte populations.
Documented Applications
Treatment and prevention of cancers such as multiple myeloma, acute myeloid leukemia, lymphoma, breast, colorectal, gastric, ovarian, lung, prostate, and non-small cell lung cancer.
Treatment and prevention of chronic viral infections including HIV, CMV, HSV, EBV, and HBV by activating and expanding virus-specific memory T cells.
Use as T cell engagers recruiting and activating T cells via CD28 and CD3 to tumor or infected cells expressing CD38, HER2 or HIV antigens.
In vitro methods for expanding virus-specific memory T cells and T cells ex vivo or in vitro using the trispecific binding proteins.
Combination therapy with standard anti-retroviral therapy for treating HIV/AIDS.
Pharmaceutical compositions comprising the trispecific binding proteins for human therapeutic use.
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