The present invention concerns polypeptides comprising a variant Fc region. More particularly, the present invention concerns Fc region-containing polypeptides that have altered effector function as a consequence of one or more amino acid modifications in the Fc region thereof.

Polypeptide variants with altered effector function

The anti-Tac monoclonal antibody is known to bind to the p55 chain of the human interleukin 2 receptor and to inhibit proliferation of T cells by blocking interleukin 2 binding. However, use of anti-Tac as an immunosuppressant drug would be impaired by the human immune response against this murine antibody. We have therefore constructed a "humanized" antibody by combining the complementarity-determining regions (CDRs) of the anti-Tac antibody with human framework and constant regions. The human framework regions were chosen to maximize homology with the anti-Tac antibody sequence. In addition, a computer model of murine anti-Tac was used to identify several amino acids which, while outside the CDRs, are likely to interact with the CDRs or antigen. These mouse amino acids were also retained in the humanized antibody. The humanized anti-Tac antibody has an affinity for p55 of 3 x 10(9) M-1, about 1/3 that of murine anti-Tac.

https://www.pnas.org/content/pnas/86/24/10029.full.pdf

A humanized antibody that binds to the interleukin 2 receptor.

A chimeric antibody with human constant region and murine variable region, having specificity to a 35 kDA polypeptide (Bp35(CD20)) expressed on the surface of human B cells, methods of production, and uses.

Chimeric antibody with specificity to human b cell surface antigen

Modular assembly of antibody genes, antibodies prepared thereby and use.

Construction and initial characterization of a mouse-human chimeric anti-TNF antibody

Chimeric antibodies were constructed in which the murine variable region of anti-colorectal cancer monoclonal antibody CO17-1A was joined with human gamma 1, gamma 2, gamma 3, and gamma 4 constant regions Human-mouse chimeric proteins were compared with the parental murine IgG2a antibody CO17-1A for their ability to participate in tumor-cell destruction by human and murine effector cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays All of the chimeric antibodies showed different degrees of ADCC with human lymphocytes, monocytes, and granulocytes and with murine macrophages Monocytes and macrophages were able to utilize the chimeric IgG1 and, to a lesser degree, IgG4 and IgG3 antibodies to lyse tumor-cell targets in ADCC assays The chimeric IgG1 and IgG4 antibodies were nearly as effective as the parental CO17-1A antibody in inhibiting tumor growth in nude mice These data indicate that chimeric IgG1 antibody is superior in its antitumor activity

https://www.pnas.org/content/pnas/85/13/4852.full.pdf

Biological activity of human-mouse IgG1, IgG2, IgG3, and IgG4 chimeric monoclonal antibodies with antitumor specificity

We have cloned the genomic DNA fragments encoding the heavy and light chain variable regions of monoclonal antibody 17-1A, and we have inserted them into mammalian expression vectors containing genomic DNA segments encoding human gamma 3 and kappa constant regions The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2/0 mouse myeloma cells resulted in the production of functional IgG that retained the specific binding to the surface antigen 17-1A expressed on colorectal carcinoma cells

Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-1A.

A chimeric protein comprising a protein having an affinity for a biological target linked through a peptide linkage to a metal binding protein or functional domain thereof not normally associated with the protein are described. The protein can be an immunoglobulin specific towards tumor or profileration associated antigens or specific towards markers of cardiovascular disease. The chimeric proteins of the present invention are useful in in vivo diagnostic techniques such as immunoscintigraphy and also as an immunotherapeutic agent.

Chimeric proteins incorporating a metal binding protein

A chimeric antibody was constructed in which the murine H- and L-chain variable regions of mAb 17-1A, raised against human colorectal cancer cells, were joined with the human constant μ and κ regions. Transfection of these constructs into the murine myeloma Sp2/0 resulted in the expression and secretion of a pentameric Ig, designated chimeric 17-1A IgM. The chimeric 17-1A IgM was subsequently compared to a previously described chimeric 17-1A IgG1 for biological activities. Both chimeric mAbs were equally effective (weight basis) in competing against the binding of murine125I-17-1A to cultures of HT-29 colon carcinoma cells. The calculated association constants for the chimeric 17-1A IgM and IgG1 were 1.63 × 108 1/mol and 3.41 × 107 1/mol, respectively. Unlike chimeric 17-1A IgG1, the chimeric 17-1A IgM was able to render colon carcinoma target cells susceptible to lysis by both xenogeneic (rabbit) and human complement. The extent of complement-mediated lysis dependent upon chimeric 17-1A IgM was correlated to 17-1A antigen expression on target cells. HT-29 colon carcinoma cells treated with chimeric 17-1A IgM did not directly result in antibody-dependent cellular cytotoxicity by human peripheral blood monocytes. However, chimeric 17-1A IgM greatly enhanced the deposition of C3 on complementtreated HT-29 cells, and concomitant incubation with monocytes resulted in heightened lysis of the tumor cells. The feasibility of enhancing host defense against gastrointestinal malignancies by the administration of this chimeric 17-1A IgM may have certain clinical advantages.

Biological characterization of a chimeric mouse-human IgM antibody directed against the 17-1A antigen.

Murine monoclonal antibody (MAb) G3-519 has been shown to recognize a conserved neutralizing epitope in the fourth constant (C4) region of the external glycoprotein gp120 of HIV-1. Inasmuch as this antibody effectively neutralized the infectivity of diverse HIV-1 isolates, it has been selected to be developed for passive immunization against HIV-1 infection in humans. In order to minimize the problem of immunogenicity of murine antibodies and to confer additional accessory immune functions, we have constructed mouse/human chimeric and humanized forms of the antibody. The chimeric antibody was constructed by cloning the murine variable regions and replacing the mouse constant regions with those from human Ig gamma 1,kappa. The humanized antibody was constructed using the human KAS variable region framework sequences as template. Engineering was guided by a three dimensional model of the murine variable region. The murine, chimeric and humanized forms of the antibody exhibited similar reactivity with the peptidic antigen in ELISA, and comparably neutralized the infectivity of HIV-1 in vitro. Taken together, our results show that the chimeric and humanized forms of G3-519 essentially retain the binding activity of the mouse parental antibody. Clinical development is planned to assess the prophylactic and therapeutic usefulness of these reshaped antibodies in humans.

Lee K. Sun

Papers

Biological activity of human-mouse IgG1, IgG2, IgG3, and IgG4 chimeric monoclonal antibodies with antitumor specificity

Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-1A.

Chimeric proteins incorporating a metal binding protein

Biological characterization of a chimeric mouse-human IgM antibody directed against the 17-1A antigen.

Construction and characterization of chimeric and humanized forms of a broadly neutralizing monoclonal antibody to HIV-1.