Link:  Pharm/Biotech Resources

Title:  Monoclonal antibodies with reduced immunogenicity

United States Patent:  6,936,698

Issued:  August 30, 2005

Inventors:  Taylor; Alexander H. (Exton, PA)

Assignee:  SmithKline Beecham (Philadelphia, PA)

Appl. No.:  905243

Filed:  July 16, 2001

Antibodies having reduced immunogenicity and methods for making them are disclosed.

SUMMARY OF THE INVENTION

One aspect of the present invention is an antibody comprising donor CDRs derived from an antigen-specific donor antibody of a non-human species and acceptor framework residues derived from a non-human primate.

Another aspect of the invention is a method for making an antibody having reduced immunogenicity in humans comprising grafting CDRs from antigen-specific non-human antibodies onto homologous non-human primate acceptor frameworks.

Another aspect of the invention is a chimpanzee VH acceptor framework I, II and III comprising an amino acid sequence as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17 or 18.

Another aspect of the invention is a chimpanzee VH acceptor framework IV comprising an amino acid sequence as set forth in SEQ ID NOs: 81, 82, 83, 84 or 85.

Another aspect of the invention is a chimpanzee Vκ acceptor framework I, II and III comprising an amino acid sequence as set forth in SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35 or 36.

Another aspect of the invention is a chimpanzee Vκ acceptor framework IV comprising an amino acid sequence as set forth in SEQ ID NOs: 86 or 87.

Another aspect of the invention is a cynomolgus VH acceptor framework I, II and III comprising an amino acid sequence as set forth in SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51 or 52.

Another aspect of the invention is a cynomolgus VH acceptor framework IV comprising an amino acid sequence as set forth in SEQ ID NOs: 88, 89, 90, 91, 92 or 93.

Another aspect of the invention is a cynomolgus Vκ acceptor framework I, II and III comprising an amino acid sequence as set forth in SEQ ID NOs: 59, 60, 61, 62, 63 or 64.

Another aspect of the invention is a cynomolgus Vκ acceptor framework IV comprising an amino acid sequence as set forth in SEQ ID NOs: 94, 95 or 96.

Yet another aspect of the invention is an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, 18, 28, 29, 30, 31, 32, 33, 34, 35 or 36.

Yet another aspect of the invention is an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NOs: 81, 82, 83, 84, 85, 86 or 87.

Yet another aspect of the invention is an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 59, 60, 61, 62, 63 or 64.

Yet another aspect of the invention is an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NOs: 88, 89, 90, 91, 92, 93, 94, 95 or 96.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

The molecular genetic aspects of antibody structure have been reviewed by S. Tonegawa in Nature 302:575-581 (1983). Briefly, antibodies are heterodimers comprised of at least two heavy and two light chains. The N-terminal domain of each heavy and light chain, termed VH and VL, respectively, fold together to form the antigen combining site. On the genetic level, the VL domain is encoded by two different gene segments, termed Vκ or Vl, and Jκ or Jl that join together to form one continuous VL region. Similarly, the VH domain is encoded by three gene segments, VH, DH, and JH, that join together to form one continuous VH region. Thus different VL and VH regions may be encoded by different combinations of Vκ or Vl, Jκ or Jl and VH, DH, and JH. This combinatorial diversity is in part the means by which the immune response generates the myriad diversity of different antibody molecules and their associated antigen specificities.

On the protein level, each heavy and light V region domain may be further divided into three CDRs. Three heavy and three light chain CDRs fold together to form the antigen binding surface and part of the underlying support structures that are required to maintain the exact three-dimensional structure of the antigen combining site. Flanking each CDR are framework regions that in most cases do not directly interact with the specific antigen, but rather serve to form the scaffold which supports the antigen binding properties of the CDRs. Each heavy and light chain has four framework regions, three derived from the VH or VL gene segment, the fourth is derived from the JH, Jκ, or Jl gene segment. Thus, the order of frameworks and CDEs from the N-terminus is framework I, CDRI, framework II, CDRII, framework III, CDRIII, framework IV. On the genetic level, all of framework I through Framework III is encoded by the V region gene segment; CDRIII is encoded jointly by both the V region and J region gene segments; framework IV is encoded entirely from the J gene segment.

Methods are provided for making engineered antibodies with reduced immunogenicity in humans and primates from non-human antibodies. CDRs from antigen-specific non-human antibodies, typically of rodent origin, are grafted onto homologous non-human primate acceptor frameworks. Preferably, the non-human primate acceptor frameworks are from Old World apes. Most preferably, the Old World ape acceptor framework is from Pan troglodytes, Pan paniscus or Gorilla gorilla. Particularly preferred is the chimpanzee Pan troglodytes. Also preferred are Old World monkey acceptor frameworks. Most preferably, the Old World monkey acceptor frameworks are from the genus Macaca. Particularly preferred is the cynomolgus monkey Macaca cynomolgus.

Particularly preferred chimpanzee (Pan troglodytes) heavy chain variable region frameworks (VH) are CPVH41-12 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 10 and the framework IV amino acid sequence shown in SEQ ID NO: 83; CPVH41-1 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 11 and the framework IV amino acid sequence shown in SEQ ID NO: 85; CPVH41-4 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 12; CPVH41-7 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 13; CPVH41-8 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 14, CPVH41-9 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 15 and the framework IV amino acid sequence shown in SEQ ID NO: 81; CPVH41-10 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 16 and the framework IV amino acid sequence shown in SEQ ID NO: 82; CPVH41-18 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 17; and CPVH41-19 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 18 and the framework IV amino acid sequence shown in SEQ ID NO: 84.

Particularly preferred chimpanzee (Pan troglodytes) light chain kappa variable region frameworks (Vκ) are CPVκ46-1 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 28; CPVκ46-3 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 29; CPVκ46-4 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 30; CPVκ46-5 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 31; CPVκ46-6 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 32 and the framework IV amino acid sequence shown in SEQ ID NO: 86; CPVκ46-7 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 33 and the framework IV amino acid sequence shown in SEQ ID NO: 87; CPVκ46-8 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 34; CPVκ46-11 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 35; and CPVκ46-14 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 36.

Particularly preferred cynomolgus (Macaca cynomolgus) heavy chain variable region frameworks (VH) are CYVH2-1 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 45 and the framework IV amino acid sequence shown in SEQ ID NO: 88; CYVH2-3 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 46 and the framework IV amino acid sequence shown in SEQ ID NO: 89; CYVH2-4 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 47 and the framework IV amino acid sequence shown in SEQ ID NO: 90; CYVH2-5 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 48 and the framework IV amino acid sequence shown in SEQ ID NO: 93; CYVH2-6 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 49 and the framework IV amino acid sequence shown in SEQ ID NO: 91; CYVH2-7 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 50; CYVH2-8 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 51; and CYVH2-10 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 52 and the framework IV amino acid sequence shown in SEQ ID NO: 92.

Particularly preferred cynomolgus (Macaca cynomolgus) light chain kappa variable region frameworks (Vκ) are CYVκ4-2 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 59; CYVκ4-3 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 60 and the framework IV amino acid sequence shown in SEQ ID NO: 94; CYVκ4-5 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 61; CYVκ4-6 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 62 and the framework IV amino acid sequence shown in SEQ ID NO: 95; CYVκ4-10 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 63; and CYVκ4-11 having the framework I, II and III amino acid sequence shown in SEQ ID NO: 64 and the framework IV amino acid sequence shown in SEQ ID NO: 96.

Isolated nucleic acid molecules encoding the chimpanzee VH and Vκ acceptor framework I, II and III amino acid sequences of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, 18, 28, 29, 30, 31, 32, 33, 34, 35 or 36 and the framework IV amino acid sequences of SEQ ID NOs: 81, 82, 83, 84, 85, 86 or 87 are also part of the present invention. Further, isolated nucleic acid molecules encoding the cynomolgus VH and Vκ acceptor framework I, II and III amino acid sequences of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 59, 60, 61, 62, 63 or 64 and the framework IV amino acid sequences of SEQ ID NOs: 88, 89, 90, 91, 92, 93, 94, 95 or 96 are also part of the present invention. Nucleic acid sequences encoding functional fragments or analogs of the VH and Vκ acceptor framework amino acid sequences are also part of the present invention.

In addition to isolated nucleic acid sequences encoding VH and Vk acceptor frameworks described herein, nucleic acid sequences complementary to these framework regions are also encompassed by the present invention. Useful DNA sequences include those sequences which hybridize under stringent hybridization conditions to the DNA sequences. See, T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982), pp. 387-389. An example of one such stringent hybridization condition is hybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for one hour. Alternatively, an exemplary stringent hybridization condition is 50% formamide, 4×SSC at 42° C. Preferably, these hybridizing DNA sequences are at least about 18 nucleotides in length.

Suitable frameworks are selected by computer homology searching among members of a database of Old World ape or monkey VH and VL regions. The framework portions of primate antibodies are useful as components of therapeutic antibodies. Moreover, primate antibody frameworks will be tolerated when used in the treatment of humans due to the close sequence homology between the genes of the primates and humans. Thus, the present invention provides for the grafting of CDRs from an antigen specific non-human donor antibody to acceptor V regions derived from non-human primate species.

The antigen specificity and binding kinetics of the donor antibody, which may be of rodent or any other non-human origin, are best preserved by selecting primate acceptor V regions that are determined by computer homology searching to be most similar to the donor antibody. Alternatively, the acceptor antibody may be a consensus sequence generated from primate V region subfamilies, or portions thereof, displaying the highest homology to the donor antibody.

The resulting engineered constructs, in which the donor CDRs are grafted onto primate acceptor frameworks, are subsequently refined by analysis of three-dimensional models based on known antibody crystal structures as found, e.g., in the Protein Data Bank (PDB), which is operated by Rutgers, The State University of New Jersey; the San Diego Supercomputer Center at the University of California, San Diego; and the National Institute of Standards and Technology-three members of the Research Collaboratory for Structural Bioinformatics (RCSB) or a similar data bank containing three-dimensional protein structures. Alternatively, computer generated three-dimensional models of the donor antibody may be computed by means of commercially available software such as "AbM" (Oxford Molecular, Oxford, UK).

Structural analysis of these models may reveal donor framework residues that are CDR-contacting residues and that are seen to be important in the presentation of CDR loops, and therefore binding avidity. A CDR-contacting residue is one which is seen in three-dimensional models to come within the van der Waals radius of a CDR residue, or could interact with a CDR residue via a salt bridge or by hydrophobic interaction. Such donor framework (CDR-contacting) residues may be retained in the engineered construct.

The modeling experiments can also reveal which framework residues are largely exposed to the solvent environment. The engineered constructs may be further improved by substituting some or all of these solvent-accessible amino acid residues with those found at the same position among human V regions most homologous to the engineered construct as disclosed in U.S. Pat. No. 5,639,641.

The engineered V regions are then joined to one or more different human or Old World ape constant regions depending on the desired secondary immune functions such as complement fixation or Fc receptor binding. Human constant regions can be selected from human immunoglobulin classes and isotypes, such as IgG (subtypes 1 through 4), IgM, IgA, and IgE. An IgG4 subtype variant containing the mutations S228P and L235E (PE mutation) in the heavy chain constant region which results in reduced effector function can also be selected. See U.S. Pat. Nos. 5,624,821 and 5,648,260.

The complete heavy and light chain genes are transferred to suitable expression vectors and co-expressed in the appropriate host cells such as chinese hamster ovary, COS or myeloma cells. The resulting engineered antibody is expected to be of substantially reduced immunogenicity when administered to humans, and to retain full binding affinity for antigen.

Acceptor V regions can be isolated specifically for each donor V region by directed PCR methodology where a non-human primate cDNA library is surveyed for acceptor frameworks most similar to the donor antibody. Oligonucleotide PCR primers homologous to the donor antibody framework I (paired with C-region 3′ PCR primers) are used to direct PCR amplification of a non-human primate, e.g., chimpanzee lymphocyte cDNA library. This would select for V-regions with framework I regions similar to the donor antibody, and sequence analysis of the obtained clones would reveal the associated framework II and III (and IV) sequences. 3′ PCR primers would then be designed based on the knowledge of the non-human primate framework III sequences thus obtained, and used to direct PCR amplification of the original cDNA library together with a vector-specific 5′ PCR primer. cDNA clones obtained from the second round of PCR amplification would have framework I and III sequences most similar to the donor antibody, and the framework II sequences would display a similar degree of sequence homology.
 

Claim 1 of 14 Claims

1. An antibody comprising:

a) a variable region comprising six complementarity determining regions (CDRs) from an antigen-specific donor antibody of a rodent and acceptor framework comprising amino acid residues from an Old World Ape, wherein at least one CDR-contacting amino acid residue from the acceptor framework is replaced with a corresponding residue from the donor framework, and wherein said CDR-contacting residue contacts a CDR residue within said antibody by the group selected from coming within the van der Waals radius of said CDR residue, a salt bridge and a hydrophobic interaction; and

b) at least one constant region from human, and

wherein said antibody has a specific binding avidity that is within about three-fold of the specific binding avidity of said antigen-specific donor antibody.

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