Humanized complement 5A receptor 1 antibodies and methods of use thereof
Inventors
Viswanathan, Karthik • Booth, Brian • Ramakrishnan, Boopathy • Wollacott, Andrew • Babcock, Gregory • Shriver, Zachary • Olinski, Lauren
Assignees
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Publication Number
US-12227587-B2
Publication Date
2025-02-18
Expiration Date
Abstract
The present disclosure provides, among other things, two different formats of humanized antibodies against human complement component 5a receptor I. The disclosure also provides a method of treating a subject having dysfunctions of C5a/C5aR1 axis pathway, including but not limited to ANCA-associated vasculitis, comprising administering to the subject in need thereof an effective amount of antibody or a nucleic encoding an antibodies binding to C5aR1 described herein, and wherein administering results in a decrease in symptoms associated with C5a/C5aR1 associated dysfunction in the subject.
Core Innovation
The invention provides humanized monospecific and biparatopic anti-C5aR1 antibodies and antigen binding fragments that bind complement component 5a receptor 1 (C5aR1). The antibodies include a VH region comprising three heavy chain complementarity determining regions defined by specific SEQ ID numbers and a VL region comprising three light chain complementarity determining regions defined by specific SEQ ID numbers.
The invention further specifies antibody formats directed to C5aR1 epitopes, including monospecific targeting of either Site I or Site II and biparatopic/tetravalent targeting that binds both Site I and Site II. The described antibodies show high C5aR1 binding affinity and reduced cross-reactivity to C5aR2 and other GPCRs.
Fc engineering is described to silence effector functions, reduce Fc-mediated effector functions, and prevent Fab arm exchange, including IgG1 Fc and IgG4 Fc formats with representative mutations such as S228P and IgG4 Fc mutations including F234V/L235E/D265G. Thermal stability enhancing modifications are also described through cysteine introduction at specified positions, and functional readouts include α signaling, calcium signaling, β-arrestin signaling or recruitment, neutrophil chemotaxis, CD11b expression, and ROS production.
Claims Coverage
The provided claims center on producing a C5aR1-binding antibody or antigen binding fragment in a host cell, with the resulting molecule defined by sequence-specific VH and VL regions. Across the claims, the inventive features include host-cell production, defined VH and VL CDR sequences, VH and VL sequence identity or exact sequence assignments, inclusion of an Fc region, and modification of the VH or VL to enhance stability.
Host-cell culturing to produce a C5aR1-binding antibody
A method of making an antibody or antigen binding fragment thereof that binds C5aR1, comprising culturing a host cell comprising a nucleic acid encoding the C5aR1 antibody or antigen binding fragment thereof and culturing the cell under conditions that allow production of the antibody or antigen binding fragment thereof.
Vh with defined hcdr sequences
The C5aR1 antibody or antigen binding fragment thereof comprises a VH region, wherein the VH comprises three HCDRs, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively.
Vl with defined lcdr sequences
The antibody or antigen binding fragment thereof further comprises a VL region, wherein the VL comprises three LCDRs, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, respectively.
VH sequence identity constraint relative to a reference sequence
The method further limits that the VH contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 14.
VL sequence identity constraint relative to a reference sequence
The method further limits that the VL includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 25.
Exact VH and VL sequence assignments
The method is defined such that the VH and VL each comprise specific amino acid sequences, with VH as SEQ ID NO: 14 and VL as SEQ ID NO: 25.
Fc region inclusion in the antibody or antigen binding fragment
The method specifies that the antibody or its antigen-binding fragment further includes an Fc region.
Stability enhancement by modifying the VH or VL
The method includes modifying the VH or VL to enhance the stability of the molecule.
Coverage is anchored on producing a C5aR1-binding antibody or antigen binding fragment in a host cell and ensuring the resulting molecule contains a VH region with defined HCDR sequences and a VL region with defined LCDR sequences. Additional claim coverage includes VH and VL sequence identity thresholds or exact VH/VL assignments, Fc region inclusion, and VH or VL modification to enhance stability.
Stated Advantages
Thermal stability enhancement is described through increased Tm thresholds.
Reduced ADCC, reduced ADCP, and reduced phagocytosis, or ablated corresponding activities, are described.
Silencing effector functions and preventing Fab arm exchange are described.
Reduced cross-reactivity to C5aR2 and other GPCRs.
High C5aR1 binding affinity.
Functional antagonism of C5a/C5aR1 axis signaling readouts, including α signaling, calcium signaling, neutrophil chemotaxis, CD11b expression, β-arrestin signaling, and ROS production.
In vivo efficacy is described, including neutropenia inhibition and stroke infarct volume protection.
Documented Applications
In vivo inhibition of neutropenia is described in squirrel monkeys and in human C5aR1 transgenic mice.
In vivo stroke protection is described as infarct volume protection in a stroke model (tMCAO).
Functional evaluation of C5aR1 pathway activity is described using assays including α signaling, calcium signaling, β-arrestin recruitment or signaling, neutrophil chemotaxis, CD11b expression, and ROS production, including ANCA-induced ROS.
Treatment concepts for C5a/C5aR1 axis dysfunction including ANCA-associated vasculitis, lupus, rheumatoid arthritis, and sepsis.
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