`
`VERSION* Per
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC1)
`wo 90/07861
`
`(51) International Patent Qassification:
`C12P 21/00, C12N 5/10, 7101, 15/00 *
`
`At
`
`(11) International Publication Number:
`
`(43) International Publication Date:
`
`26 July 1990 (26.07.90)
`
`(21) International Application Number:
`
`PCT/US89/05857
`
`(22) International Filing Date:
`
`28 December 1989 (28.12.89)
`
`(30) Priority data:
`290,975
`310,252
`
`28 December 1988 (28.12.88) US
`13 February 1989 (13.02.89) US
`
`(71)Applicant: PROTEIN DESIGN LABS, INC. [US/US];
`3181 Porter Drive, Palo Alto, CA 94304 (US).
`
`(72) Inventors: QUEEN, Cary, L. ; 1300 Oak Creek Drive, Palo
`Alto, CA 94304 (US). SELICK, Harold, Edwin ; 1673
`Sunnyslope Avenue, Belmont, CA 94002 (US).
`
`(74)Agent: SMITH, William, M.; Townsend and Townsend,
`One Market Plaza, 2000 Steuart Tower, San Francisco,
`CA 94105 (US).
`
`(81) Designated States: AT, AT (European patent), AU, BB, BE
`(European patent), BF (OAPI patent), BG, BJ (OAPI
`patent), BR, CF (OAPI patent), CG (OAPI patent), CH,
`CH (European patent), CM (OAPI patent), DE, DE
`(European patent), OK, ES (European patent), Fl, FR
`(European patent), GA (OAPI patent), GB, GB (Euro(cid:173)
`pean patent), HU, IT (European patent), JP, KP, KR,
`LK, LU, LU (European patent), MC, MG, ML (OAPI
`patent), MR (OAPI patent), MW, NL, NL (European
`patent), NO, RO, SD, SE, SE (European patent), SN
`(OAPI patent), SU, TD (OAPI patent), TG (OAPI pa(cid:173)
`tent).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: .CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR p55 TAC PROTEIN OF THE IL-2 RECEPTOR
`
`(57) Abstract
`
`Novel methods for designing humanized immunoglobulins having one or more complementary determining regions
`(CDR's) from a donor immunoglobulin and a framework region from a human immunoglobulin comprising first comparing the
`framework or variable region amino acid sequence of the donor immunoglobulin to corresponding sequences in a collection of
`human immunoglobulin chains, and selecting as the human immunoglobulin one of the more homologous sequences from the
`collection. Each humanized immunoglobulin chain may comprise about 3 or more amino acids from the donor immunoglobulin
`in addition to the CDR's, usually at least one of which is immediately adjacent to a CDR in the donor immunoglobulin. The
`heavy and light chains may each be designed by using any one or all three additional position criteria. When combined into an
`intact antibody, the humanized immunoglobulins of the present invention will be substantially non-immunogenic in humans and
`retain substantially the same affinity as the donor immunoglobulin to the antigen, such as a protein or other compound contain(cid:173)
`ing an epitope.
`
`\ '
`
`* (Referred to in Per Oilll.ette No. 2211990, Section II)
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identifY States pany to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT Austria
`AU Ausaalia
`B8
`Batbldos
`BE
`BcJsi,lm
`Burkina FISID
`BF
`8u4aria
`BG
`BJ
`Benin
`BR
`Brazil
`CA
`Canada
`CF
`Central Aftican Republic
`CG
`Congo
`01
`Switzerland
`CM Cameroon
`DE Germany, Federal Rcpubfac of
`DK Dcnmatk
`
`m
`Spain
`FT
`Fmland
`FR
`France
`GA Gabon
`GB United KiJiadom
`HU HUDJIIY
`rr
`Italy
`Japan
`JP
`KP Democratic People's Republic
`of Korea
`KR Republic of Korea
`u
`LicchtcDSlCin
`LK
`Sril..ania
`w Luxembourg
`MC Monaa:t
`
`MG Madapsc:ar
`ML Mali
`MR Mauritania
`Pr'Nl Malawi
`NL Netherlands
`1'10 Norway
`Romania
`RO
`Sudan
`Sl
`Sweden
`SE
`Sene sal
`S'll
`Soviet Union
`SJ
`TD
`Chad
`TG
`T010
`us
`United States of America
`
`_..,:
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`J
`-,.
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`i!!
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`CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR p55 TAC PROTEIN
`OF THE IL-2 RECEPTOR
`
`Field of the Invention
`The present invention relates generally to the
`combination of recombinant DNA and monoclonal antibody
`technologies for developing novel therapeutic agents and,
`more particularly, to the production of non-immunogenic
`antibodies and their uses.
`
`Background of the Invention
`In mammals, the immune response is mediated by two
`types of cells that interact specifically with foreign
`material, i.e., antigens. One of these cell types, B-cells,
`are responsible for the production of antibodies. The second
`cell class, T-cells, include a wide variety of cellular
`subsets controlling the in vivo function of both B-cells and
`a wide variety of other hematopoietic cells, including T(cid:173)
`cells.
`
`One way in which T-cells exert this control is
`through the production of a lymphokine known as interleukin-2
`(IL-2), originally named T-cell growth factor.
`IL-2's prime
`function appears to be the stimulation and maintenance of T(cid:173)
`cells.
`Indeed, some immunologists believe that IL-2 may be
`at the center of the entire immune response (~,Farrar, J.,
`et al., Immunol. Rev. £1:129-166 (1982), which is
`incorporated herein by reference).
`To exert its biological effects, IL-2 interacts
`with a specific high-affinity membrane receptor (Greene, w.,
`et al., Progress in Hematology XIV, E. Brown, Ed., Grune and
`Statton, New York (1986), at pgs. 283 ff). The human IL-2
`receptor is a complex .multichain glycoprotein, with one
`chain, known as the Tac peptide, being about 55kD in size
`(~, Leonard, W., et al., J. Biol. Chem. 260:1872 (1985),
`which is incorporated herein by reference). A gene encoding
`this protein has been isolated, and predicts a 272 amino acid
`peptide, including a 21 amino acid signal peptide (~,
`Leonard, w., et al., Nature 311: 626 (1984)). The 219 NH
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`terminal amino acids of the p55 Tao protein apparently
`comprise an extracellular domain (~, Leonard, w., et al.,
`Science, ~:633-639 (1985), which is incorporated herein by
`reference).
`Much of the elucidation of the human IL-2
`receptor's structure and function is due to the development
`of specifically reactive monoclonal antibodies.
`In
`particular, one mouse monoclonal antibody, known as anti-Tac
`(Uchiyama, et al., J. Immunol. 126:1393 (1981)) has shown
`that IL-2 receptors can be detected on T-cells, but also on
`cells of the monocyte-macrophage family, Kupffer cells of the
`liver, Langerhans• cells of the skin and, of course,
`activated T-cells.
`Importantly, resting T-cells, B-cells or
`circulating machrophages typically do not display the IL-2
`receptor (Herrmann, et al., J. EzP. Med. 162:1111 (1985)).
`The anti-Tao monoclonal antibody has also been used
`to define lymphocyte functions that require IL-2 interaction,
`and has been shown to inhibit various T-cell functions,
`including the generation of cytotoxic and suppressor T
`lymphocytes in cell culture. Also, based on studies with
`anti-Tao and other antibodies, a variety of disorders are now
`associated with improper IL-2 receptor expression by T-cells,
`in particular adult T-cell leukemia.
`More recently, the IL-2 receptor has been shown to
`be an ideal target for novel therapeutic approaches to T-cell
`mediated diseases. It has been proposed that IL-2 receptor
`specific antibodies, such as the anti-Tao monoclonal
`antibody, can be used either alone or as an immunoconjugate
`(~, with Ricin A, isotopes and the like) to effectively
`remove cells bearing the IL-2 receptor. These agents can,
`for example, theoretically eliminate IL-2 receptor-expressing
`leukemic cells, certain B-cells, or activated T-cells
`involved in a disease state, yet allow the retention of
`mature normal T-cells and their precursors to ensure the
`capability of mounting a normal T-cell immune response as
`needed.
`In general, most other T-cell specific agents can
`destroy essentially all peripheral T-cells, which limits the
`agents• therapeutic efficacy. overall, the use of
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`appropriate monoclonal antibodies specific for the IL-2
`receptor may have therapeutic utility .in autoimmune diseases,
`organ transplantation and. any unwanted response by activated
`T-cells.
`Indeed, clinical trials have been initiated using,
`5 ~, anti-Tac antibodies (~, generally, Waldman, T., et
`al., Cancer Res. 45:625 (1985) and Waldman, T., Science
`232:727-732 (1986), both of which are incorporated herein by
`reference).
`Unfortunately, the use of the anti-Tac and other
`non-human monoclonal antibodies have certain drawbacks,
`particularly in repeated therapeutic regimens as explained
`below. Mouse monoclonal antibodies, for example, do not fix
`human complement well, and lack other important
`immunoglobulin functional characteristics when used in
`humans.
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`Perhaps more importantly, anti-Tac and other non(cid:173)
`human monoclonal antibodies contain substantial stretches of
`amino acid sequences that will be immunogenic when injected
`into a human patient. Numerous studies have shown that,
`after injection of a foreign antibody, the immune response
`elicited by a patient against an antibody can be quite
`strong, essentially eliminating the antibody's therapeutic
`utility after an initial treatment. Moreover, as increasing
`numbers of different mouse or other antigenic (to humans)
`monoclonal antibodies can be expected to be developed to
`treat various diseases, after the first and second treatments
`with. any different non-human antibodies, subsequent
`treatments even for unrelated therapies can be ineffective or
`even dangerous in themselves.
`While the production of so-called "chimeric
`antibodies" (~, mouse variable regions joined to human
`constant regions) has proven somewhat successful, a
`significant immunogenicity problem remains.
`In general, the
`production of human immunoglobulins reactive with the human
`IL-2 receptor, as with many human antigens, has been
`extremely difficult using typical human monoclonal antibody
`production techniques. Similarly, utilizing recombinant DNA
`technology to produce so-called "humanized" antibcdies (see,
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`~, EPO Publication No. 0239400), provides uncertain
`results, in part due to unpredictable binding affinities.
`Thus, there is a need for improved forms of human(cid:173)
`like immunoglobulins, such as those specific for the human
`IL-2 receptor, that are substantially non-immunogenic in
`humans, yet easily and economically produced in a manner
`suitable for therapeutic formulation and other uses. The
`present invention fulfills these and other needs.
`
`summary of the Invention
`The present invention provides novel compositions
`useful, for example, in the treatment of T-cell mediated
`human disorders, the compositions containing human-like
`immunoglobulins specifically capable of blocking the binding
`of human IL-2 to its receptor andjor capable of binding to
`the p55 Tac protein on human IL-2 receptors. The
`immunoglobulins can have two pairs of light chainjheavy chain
`complexes, typically at least one pair having chains
`comprising mouse complementarity determining regions
`functionally joined to human framework region segments. For
`example, mouse complementarity determining regions, with or
`without additional naturally-associated mouse amino acid
`residues, can be used to produce human-like antibodies
`capable of binding to the human IL-2 receptor at affinity
`levels stronger than about 10 8 M-1
`•
`The immunoglobulins, including binding fragments
`and other derivatives thereof, of the present invention may
`be produced readily by a variety of recombinant DNA
`techniques, with ultimate expression in transfected cells,
`preferably immortalized eukaryotic cells, such as myeloma or
`hybridoma cells. Polynucleotides comprising a first sequence
`coding for human-like immunoglobulin framework regions and a
`second sequence set coding for the desired immunoglobulin
`complementarity determining regions can be produced
`synthetically or by combining appropriate eDNA and genomic
`DNA segments.
`The human-like immunoglobulins may be utilized
`alone in substantially pure form, or complexed with a
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`cytotoxic agent, such as a radionuclide, a ribosomal
`inhibiting protein or a cytotoxic agent active at cell
`surfaces. All of these compounds will be particularly useful
`in treating T-cell mediated disorders. The human-like
`immunoglobulins or their complexes can be prepared in a
`pharmaceutically accepted dosage form, which will vary
`depending on. the mode of administration.
`The present invention also provides novel methods
`for designing human-like immunoglobulin chains having one or
`more complementarity determining regions (CDR's) from a donor
`immunoglobulin and a framework region from a human
`immunoglobulin, the preferred methods comprising first
`comparing the framework or variable region amino acid
`sequence of the donor immunoglobulin to corresponding
`sequences in a collection of human immunoglobulin chains, and
`selecting as the human immunoglobulin one of the more
`homologous sequences from the collection. The human
`immunoglobulin, or acceptor immunoglobulin, sequence is
`typically s.elected from a collection of at least 10 to 20
`immunoglobulin chain sequences, and usually will have the
`highest homology to the donor immunoglobulin sequence of any
`s.equence in the collection. The human immunoglobulin
`framework sequence will typically have about 65 to 70%
`homology or more to the donor immunoglobulin framework
`sequences. The donor immunoglobulin may be either a heavy
`chain or light chain (or both), and the human collection will
`contain the same kind of chain. A humanized light and heavy
`chain can be used to form a complete humanized immunoglobulin
`or antibody, having two light/heavy chain pairs, with or
`without partial or full-length human constant regions and
`other proteins.
`In another embodiment of the present invention,
`either in conjunction with the above comparison step or
`separately, additional amino acids in an acceptor
`immunoglobulin chain may be replaced with amino acids form
`the CDR-donor immunoglobulin chain. More specifically,
`further optional substitutions of a human framework amino
`acid of the acceptor immunoglobulin with a corresponding
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`amino acid from a donor immunoglobulin will be made at
`positions in the immunoglobulins where:
`the amino acid in the human framework region
`(a)
`of an acceptor immunoglobulin is rare for that position and
`the corresponding amino acid in the donor immunoglobulin is
`common for that position in human immunoglobulin sequences:
`or
`
`the amino acid is immediately adjacent to one
`(b)
`of the CDR's; or
`(c) the amino acid is predicted to be within about
`3A of the CDR's in a three-dimensional immunoglobulin model
`and capable of interacting with the antigen or with the CDR's
`of the humaniz.ed immunoglobulin.
`The humanized immunoglobulin chain will typically
`comprise at least about 3 amino acids from the donor
`immunoglobulin in addition to the CDR's, usually at least one
`of which is immediately adjacent to a CDR in the donor
`immunoglobulin. The heavy and light chains may each be
`designed by using any one or all three of the position
`criteria.
`
`When combined into an intact antibody, the
`humanized light and heavy chains of the present invention
`will be substantially non-immunogenic in humans and retain
`substantially the same affinity as the donor immunoglobulin
`to the antigen (such as a protein or other compound
`containing an epitope). These affinity levels can vary from
`about 108 M-1 or higher, and may be within about 4 fold of the
`donor immunoglobulin's original affinity to the antigen.
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`BRIEF DESCRIPTION OF THE FIGURES
`Figure 1. Comparison of sequences of anti-Tao
`heavy chain (upper lines) and Eu heavy chain (lower lines).
`The 1-letter code for amino acids is used. The first amino
`acid on each line is numbered at the left.
`Identical amino
`acids in the two sequences are connected by lines. The 3
`CDRs are underlined. Other amino acid positions for which
`the anti-Tao amino acid rather than the Eu amino acid was
`used in the humanized anti-Tao heavy chain are denoted by
`an *·
`
`Figure 2. Comparison of sequences of anti-Tao
`light chain (upper lines) and Eu light chain (lower lines).
`The single-letter code for amino acids is used. The first
`amino acid on each line is numbered at the left.
`Identical
`amino acids in the two sequences are connected by lines. The
`3 CDRs are underlined. Other amino acid positions for which
`the anti-Tao amino acid rather than the Eu amino acid was
`used in the humanized anti-Tao heavy chain are denoted by
`an *·
`
`Figure 3. Nucleotide sequence of the gene for the
`humanized anti-Tao heavy chain variable region gene. The
`translated amino acid sequence for the part of the gene
`encoding protein is shown underneath the nucleotide sequence.
`The nucleotides TCTAGA at the beginning and end of the gene
`are Xba I sites. The mature heavy chain sequence begins with
`amino acid #20 Q.
`Figure 4. Nucleotide sequence of the gene for the
`humanized anti-Tac light chain variable region gene. The
`translated amino acid sequence for the part of the gene
`encoding protein is shown underneath the nucleotide sequence.
`The nucleotides TCTAGA at the beginning and end of the gene
`are Xba I sites. The mature light chain sequence begins with
`amino acid #21 D.
`Figure 5. A. Sequences .of the four
`oligonucleotides used to synthesize the humanized anti-Tao
`heavy chain gene, printed 5 1 to 3 1 • B. Relative positions
`of the oligonucleotides. The arrows point in the 3'
`direction for each oligonucleotide.
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`(A) Sequences of the four
`Figure 6.
`oligonucleotides used to synthesize the humanized anti-Tac
`light chain gene, printed 5' to 3'.
`(B) Relative positions
`of the oligonucleotides. The arrows point in the 3'
`direction. for each oligonucleotide. The position of a Hind
`III site in the overlap of JFD2 and JFD3 is shown.
`Figure 7. Schematic diagram of the plasmid
`pHuGTAC1 used to express the humanized anti-Tac heavy chain.
`Relevant restriction sites are shown, and coding regions of
`the heavy chain are displayed as boxes. The direction of
`transcription from the immunoglobulin (Ig) promoter is shown
`EH = heavy chain enhancer, Hyg = hygromycin
`by an arrow.
`resistance gene.
`Figure 8. Schematic diagram of the plasmid pHuLTAC
`used to express the humanized anti-Tac light chain. Relevant
`restriction sites are shown, and coding regions of the light
`chain are displayed as boxes. The direction of transcription
`from the Ig promoter is shown by an arrow.
`Figure 9. Fluorocytometry of HUT-102 and Jurkat
`cells stained with anti-Tac antibody or humanized anti-Tac
`antibody followed respectively by fluorescein-conjugated goat
`anti-mous~ Ig antibody or goat anti-human Ig antibody, as
`labeled.
`In each panel, the dotted curve shows the results
`when the first antibody was omitted, and the solid curve the
`results when first and second (conjugated) antibodies were
`included as described.
`Figure 10.
`(A) Fluorocytometry of HUT-102 cells
`stained with 0-40 ng of anti-Tac as indicated, then with
`biotinylated anti-Tac, and then with phycoerythrin-conjugated
`avidin.
`(B) Fluorocytometry of HUT-102 cells stained with
`the indicated antibody, then with biotinylated anti-Tac, and
`then with phycoerythrin-conjugated avidin.
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`DETAILED DESCRIPTION OF THE INVENTION
`In accordance with one embodiment of the present
`invention, human-like immunoglobulins specifically reactive
`with desired epitopes, such as those on the IL-2 receptor on
`human T-cells, are provided. These immunoglobulins, which
`have binding affinities of at least about 108 M-1
`, and
`preferably 109 M-1 to 1010 M-1 or stronger, are capable of,
`~, blocking the binding of IL•2 to human IL-2 receptors.
`The human-like immunoglobulins will have a human-like
`framework and can have complementarity determining regions
`(CDR's) from an immunoglobulin, typically a mouse
`immunoglobulin, specifically reactive with an epitope on p55
`Tao protein. The immunoglobulins of the present invention,
`which can be produced economically in large quantities, find
`use, for example, in the treatment of T-cell mediated
`disorders in human patients by a variety of techniques.
`The basic antibody structural unit is known to
`comprise a tetramer. Each tetramer is composed of two iden(cid:173)
`tical pairs of polypeptide chains, each pair having one
`"light" (about 25kD) and one "heavy" chain (about 50-70kD).
`The NH2-terminus of each chain begins a variable region of
`about 100 to 110 or more amino acids primarily responsible
`for antigen recognition. The COOH terminus of each chain
`defines a constant region primarily responsible for effector
`function.
`
`Light chains are classified as either kappa or
`lambda. Heavy chains are classified (and subclassified) as
`gamma, mu, alpha, delta, or epsilon, and define the
`antibody's isotype as IgG, IgM, IgA, IgD and IgE,
`respectively. Within light and heavy chains, the variable
`and constant regions are joined by a "J" region of about 12
`or more amino acids, with the heavy chain also including a
`"D" region of about 12 more amino acids.
`(See, generally,
`Fundamental Immunology, Paul, w., Ed., Chapter 7, pgs. 131-
`166, Raven Press, N.Y. (1984), which is incorporated herein
`by reference.)
`The variable regions of each light/heavy chain pair
`form the antibody binding site. The chains all exhibit the
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`same general structure of relatively conserved framework
`regions joined by three hypervariable regions, also called
`CDR's (~, "Sequences of Proteins of Immunological
`Interest, 11 Kabat, E., et al., u.s. Department of Health and
`Human Services, (1983)~ and Cholthia and Lesk, J. Mol. Biol.,
`196:901-917 (1987), which are incorporated herein by
`reference). The CDR's from the two chains of each pair are
`aligned by the framework regions, enabling binding to a
`specific epitope.
`As used herein, the term "immunoglobulin" refers to
`a protein consisting of one or more polypeptides
`substantially encoded by immunoglobulin genes. The
`recognized immunoglobulin genes include the kappa, lambda,
`alpha, gamma, delta, epsilon and mu constant region genes, as
`well as the myriad immunoglobulin variable region genes. The
`immunoglobulins may exist in a variety of forms besides
`antibodies; including, for example, Fv, Fab, and F(ab) 2 , as
`well as in single chains (~, Huston, et al., Proc. Nat.
`Acad. Sci. U.S.A., 85:5879-5883 (1988) and Bird, et al.,
`Science, 242:423-426 (1988), which are incorporated herein by
`reference).
`(See, generally, Hood, et al., "Immunology",
`Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood,
`Nature, 323:15-16 (1986), which are incorporated herein by
`reference) •
`Chimeric antibodies are antibodies whose light and
`heavy chain genes have been constructed, typically by genetic
`engineering, from immunoglobulin gene segments belonging to
`different species. For example, the variable (V) segments of
`the genes from a mouse monoclonal antibody may be joined to
`human constant (C) segments, such as ~1 and ~3 • A typical
`therapeutic chimeric antibody is thus a hybrid protein
`consisting of the V or antigen-binding domain from a mouse
`antibody and the c or effector domain from a human antibody
`(~, A.T.C.C. Accession No. CRL 9688 secretes an anti-Tac
`chimeric antibody), although other mammalian species may be
`used.
`
`As used herein, the term "framework region" refers
`to those portions of immunoglobulin light and heavy chain
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`variable regions that are relatively conserved (~, other
`than the CDR •.s) among different immunoglobulins in a single
`species, as defined by Kabat, et al., ~· cit. As used
`herein, a "human-like framework region" is a framework region
`that in each existing chain comprises at least about 70 or
`more amino acid residues, typically 75 to 85 or more
`residues, identical to those in a human immunoglobulin.
`As used herein, the term "human-like
`immunoglobulin" refers to an immunoglobulin comprising a
`human-like framework and in which any constant region present
`is substantially homologous to a human immunoglobulin
`constant region, i.e., at least about 85-90%, preferably
`about 95% identical. Hence, all parts of a human-like
`immunoglobulin, except possibly the CDR's, are substantially
`homologous to corresponding parts of one or more native human
`immunoglobulin sequences. For example, a human-like
`immunoglobulin would not encompass a chimeric mouse variable
`region/human constant region antibody.
`In accordance with another general aspect of the
`present invention, also included are criteria by which a
`limited number of amino acids in the framework of a human(cid:173)
`like or h'q.manized immunoglobulin chain are chosen to be the
`same as the amino acids at those positions in the donor Ig
`rather than in the acceptqr Ig, in order to increase the
`affinity of an antibody comprising the humanized
`immunoglobulin chain.
`This aspect of the present invention is based in
`part on the model that two contributing causes of the loss of
`affinity in prior means of producing humanized antibodies
`(using as examples mouse antibodies as the source of CDR's)
`are:
`
`(1) When. the mouse COR's are combined with the
`human framework, the amino acids in the framework close to
`the CDR's becom.e human instead of mouse. Without intending
`to be bound by theory, we believe that these changed amino
`acids may slightly distort the CDR's, because they create
`different electrostatic or hydrophobic forces than in the
`donor mouse antibody, and the distorted CDR's may not make as
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`effective contacts with the antigen as the CDR's did in the
`donor antibody;
`{2) Also, amino acids in the original mouse
`antibody that are close to, but not part of, the CDR's (i.e.,
`still part of the framework), may make contacts with the
`antigen that contribute to affinity. These amino acids are
`lost when the antibody is humanized, because all framework
`amino acids are made human.
`To avoid these problems, and to produce humanized
`antibodies that have a very strong affinity for a desired
`antigen, the present invention uses the following four
`criteria for designing humanized immunoglobulins. These
`criteria may be used singly, or when necessary in
`combination, to achieve the desired affinity or other
`characteristics.
`
`Criterion I: As acceptor, use a framework from a p~rticular
`human immunoglobulin that is unusually homologous to the
`donor immunoglobulin to be humanized, or use a consensus
`framework from many human antibodies. For example,
`comparison of the sequence of a mouse heavy {or light) chain
`variable region against human heayY {or light) variable
`regions in a data bank (for example, the National Biomedical
`Research Foundation Protein Identification Resource) shows
`that the extent of homology to different human regions varies
`greatly, typically from about 40% to about 60-70%. By
`choosing as the acceptor immunoglobulin one of the human
`heavy {respectively light) chain variable regions that is
`most homologous to the heavy (respectively light) chain
`variable region of the donor immunoglobulin, fewer amino
`acids will be changed in going from the donor immunoglobulin
`to the humanized immunoglobulin. Hence, and again without
`intending to be bound by theory, it is believed that there is
`a smaller chance of changing an amino acid near the CDR's
`that distorts their conformation. Moreover, the precise
`overall shape of a humanized antibody comprising the
`humanized immunoglobulin chain may more closely resemble the
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`shape of the donor antibody, also reducing the chance of
`distorting the CDR's.
`Typically, one of the 3-5 most homologous heavy
`chain variable region sequences in a representative
`collection of at least about 10 to 20 distinct human heavy
`chains will be chosen as acceptor to provide the heavy chain
`framework, and similarly for the light chain. Preferably,
`one of the 1-3 most homologous variable regions will be used.
`The selected acceptor immunoglobulin chain will most
`preferably have at least about 65% homology in the framework
`region to the donor immunoglobulin.
`Regardless of how the acceptor immunoglobulin is
`chosen, higher affinity may be achieved by selecting a small
`number of amino acids in the framework of the humanized
`immunoglobulin chain to be the same as the amino acids at
`those positions in the donor rather than in the acceptor.
`The following criteria define what amino acids may be so
`selected. Preferably, at most or all amino acid positions
`satisfying one of these criteria, the donor amino acid will
`in fact be selected.
`
`Criterion II: If an amino acid in the framework of the human
`acceptor immunoglobulin is unusual (..L...st.t.., "rare", which as
`used herein indicates an amino acid occurring at that
`position in no more than about 10% of human heavy
`(respectively light) chain v region sequences in a
`representative data bank), and if the donor amino acid at
`that position is typical for human sequences (i.e., "common",
`which as used herein indicates an amino acid occurring in at
`least about 25% of sequences in a representative data bank),
`then the donor amino .acid rather than the acceptor may be
`selected. This criterion helps ensure that an atypical amino
`acid in the human framework does not disrupt the antibody
`structure. Moreover, by replacing an unusual amino acid with
`an amino acid from the donor antibody that happens to be
`typical for human antibodies, the humanized antibody may be
`made less immunogenic.
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`Criterion III: In the positions immediately adjacent to the
`3 CDR's in the humanized immunoglobulin chain, the donor
`amino acid rather than acceptor amino acid may be selected.
`These amino acids are particularly likely to interact with
`the amino acids in the CDR's and, if chosen from the
`acceptor, distort the donor CDR's and reduce affinity.
`Moreover, the adjacent amino acids may interact directly with
`the antigen (Amit et al., Science, 233, 747-753 (1986), which
`is incorporated herein by reference) and selecting these
`amino acids from the donor may be desirable to keep all the
`antigen contacts that provide affinity in the original
`antibody.
`
`Criterion IV: A 3-dimensional model, typically of the
`original donor antibody, shows that certain amino acids
`outside of the CDR's are close to the CDR's and have a good
`probability of interacting with amino acids in the CDR's by
`hydrogen bonding, Van der waals forces, hydrophobic
`interactions, etc. At those amino acid positions, the donor
`amino acid rather than the acceptor immunoglobulin amino acid
`may be selected. Amino acids according to this criterion will
`generally have a side chain atom within about 3 angstrom
`units of some site in the CDR's and must contain atoms that
`could interact with the CDR atoms according to established
`chemical forces, such as those listed above. Computer
`programs to create models of proteins such as antibodies are
`generally available and well known to those skilled in the
`art (~, Loew et al., Int. J. Quant. Chem., Quant. Biol.
`Symp., 15:55-66 (1988); Bruccoleri .§t. al., Nature, m,
`564-568 (1988); Chothia et al., Science, 233:755-758 (1986),
`all of which are incorporated herein by reference). These do
`not form part of the invention.
`Indeed, because a