Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70521—CD28, CD152
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to molecules which can cross-link: or cross-react with the costimulatory molecules B7.1 and B7.2 expressed on professional antigen presenting cells (APCs) leading to the inhibition of antigen-specific T cell activation.
• APCs professional antigen presenting cells
• the present invention also concerns methods to produce these molecules and use of these molecules to treat diseases, such as transplant rejection, graft versus host disease (GVHD), allergy and autoimmune diseases, where antigen- specific T cell activation is pathogenic.
• mAb's monoclonal antibodies
• mAb's monoclonal antibodies
• their suboptimal stability and affinity and their large molecular size In order to solve these problems several modified antibodies, antibody constructs and peptide and nonpeptide antigen binding fragments have been developed by bioengineering or chemical methods.
• Murine mAb's were made less antigenic to humans by CDR grafting (Winter and Harris, 1993).
• mAb's were made more effective by conjugating chemotherapeutic drugs and other toxins to the antibodies (Ghetie and Vitetta, 1994).
• bispecific antibody constructs capable of simultaneously binding two different epitopes on the same- or different antigens.
• bispecific antibodies have been produced using a variety of methods: a) antibodies of different specificities or univalent fragments of pepsin-treated antibodies of different specificities have been chemically linked (Fanger et al., 1992); b) two hybridomas secreting antibodies of different specificity have been fused and the resulting bispecific antibodies from the mixture of antibodies were subsequently isolated; c) geniticcdly engineered single chain antibodies have been used to produce non-covalently linked bispecific antibodies (e.g.
• trivalent antibody constructs named triabodies
• triabodies trivalent antibody constructs
• Phage display of Ab combinatorial libraries resulting in the production of high-affinity antibodies and screening of random DNA sequence phage display libraries for small antigen-binding peptides that mimic the antigen specific binding activity of an antibody are other approaches to produce more effective antigen-binding molecules
• Peptidic or non-peptidic molecules which are derived from 'High Throughput Screening' (HTS) of chemical or natural libraries and inhibit protein-protein interactions (Sarubbi et al., 1996) can also be used as effective antigen-binding molecules.
• HTS High Throughput Screening'
• hybridomas derived from immunized transgenic mice, containing large sections of the human immunoglobulin (Ig) gene loci in the germ line, integrated by the yeast artificial chromosomal (YAC) technology, is a method to obtain effective blocking antibodies (Mendez et al, 1997).
• TcR/CD3 complex has two functions in antigen-induced activation: a recognition function in which a specific antigen is recognized in the context of the appropriate MHC molecule, and a signaling function in which the recognition event is transmitted across the plasma membrane (Im convinced et al., 1985; Weiss and Imboden, 1987).
• a recognition function in which a specific antigen is recognized in the context of the appropriate MHC molecule
• a signaling function in which the recognition event is transmitted across the plasma membrane
• T cells require a second signal to induce proliferation and maturation into effector cells. This costimulatory signal is provided by the cell surface of APCs (Springer et al., 1987).
• T cell inactivation results in T cell inactivation in the form of clonal anergy (Mueller et al., 1989).
• the interaction of a number of accessory molecules present on the cell surface of T cells with known ligands on the APC have been implicated in providing the costimulatory signal in T cell activation: CD2 with its ligand CD58 (LFA-3), CD 11 a/CD 18 (LEA- 1 ) with CD54 (ICAM- 1 ), CD28 with B7, and CD29/CD49d 5VLA-4) with VCAM-1 (Selvaro] et al., 1987; Springer, 1990; Marlin and Springer, 1987, Van Noesel et al., 1988; Iinsley et al., 1990; Damle et al., 1991).
• CD3 Mab in the absence of APC results in anergy
• cross-linking of the CD28 molecule using ant ⁇ -CD28 Mab could prevent anergy induction.
• addition of Fab fragments of anti-CD28 Mabs caused T cell anergy
• mouse fibroblasts co-transfected with M HC-DR-7 and human B7 1 but not with ICAM- 1 , could prevent anergy induction of DR-7-specific ⁇ llore ⁇ ctive human T cells (Boussiotis et al., 1993a).
• B7 B7.1 (CD80), originally named B7/BB1, and B7.2 (CD86), originally named B70.
• B7.1 is a monomeric transmembrane glycoprotein with an apparent molecular mass of 45-65 kDa and is a member of the immunoglobulin superfamily (Freeman et al., 1989). It was initially reported that the expression of the B7.1 molecule was restricted to activated B cells (Freeman et al., 1989) and monocytes stimulated with IFN- ⁇ (Freedman et al., 1991). More recently, B7.1 expression has also been found on cultured peripheral blood dendritic cells (Young et al. 1992) and on m vitro activated T cells (Azuma et al., 1993a).
• the expression of the B7.1 molecule in a number of normal and pathological tissues has been examined by iinmunohistochemistry using the anti-B7.1 Mab B7-24 (Vandenberghe et al., 1993).
• the B7.1 molecule was shown to be constitutively expressed in vivo on dendritic cells in both lymphoid and non-lymphoid tissue.
• Monocytes/macrophages were only found to be positive under inflaminatory conditions and endothelial cells were always negative.
• the number of B7.1 positive cells in skin lesions of patients with acute GVHD was strongly increased compared to normal skin.
• B7.2 is also a transmembrane glycoprotein with an apparent molecular mass of approximately 70 kDa and is also a member of the immunoglobulin superfamily (Freeman et al., 1993; Azuma et al., 1993b).
• the B7.2 molecule seems to have a very similar histo-distribution pattern to B7.1 , with the exception that the induction of cell- surface expression seems to be faster (Freeman et al., 1993) and that it is present on freshly isolated monocytes (Azuma et al., 1993b). Also the expression of the B7.2 molecule was found to overlap to a large extent with B7.1, with minor differences in some B cell subsets in and around germinal centers, which most likely reflects different activation status of the B cells (de Boer, unpublished results).
• B7 1 has been identified on activated _B cells and activated keratinocytes (Boussiotis et al 1993b) This molecule can bind to CD28 but not CTLA-4 (see below) However this molecule does not seem to be able to stimulate T cells via CD28
• a second B7-l ⁇ ke molecule has been described on EBV- transformed B cell lines and functionally interacts with CTLA-4 on T cells resulting m apoptosis of previously activated T cells (Gribben et al 1995)
• CD28 a homodimenc transmembrane glycoprotein with an apparent molecular mass of 44 kDa and a memoer of the immunoglobulin superfamily is expressed on approximately 95% of the CD4-pos ⁇ t ⁇ ve T cells and 50% of the CD8 positive T cells (June et al 1990) CD28 regulates a signal transduction pathway distinct from that induced by the TcR/CD3 complex (Vandenberghe et al , 1992) Many studies indicate that costimulation of T cells by cross-linking the CD28 molecule with Mab results in a greatly enhanced activation, which is accompanied by the production of large amounts of ⁇ nterleukm-2 (IL 2) (Thompson et al 1989, June et al 1989) and other cytokmes Furthermore anti CD28 Mabs can be replaced by B7 the natural ligand for CD28 (Lmsley et al 1991 Gimmi et al 1991 de Boer et al 1992) The B7-CD28 interaction can result in a strong prol
• CTLA-4 and CD28 bind the B7 molecules (linsley et al., 1991; Freeman et al., 1993; Azuma et al., 1993b).
• CTLA-4 has, compared to CD28, a 10 to 20-fold higher affinity for B7 (linsley et al., 1991), and, whereas CD28 is constitutively expressed at relatively high levels on T cells, CTLA-4 is only expressed at low levels on activated T cells (Linsley et al., 1992).
• mRNA for CTLA-4 can be detected shortly after activation of T cells, cell surface expression is only found after 2-4 days in culture (Linsley et al., 1992).
• CTLA-4 The exact function of CTLA-4 has been, until recently, controversial (summarized in Science Perspective, Allison and Krummel, 1995 and Refs therein).
• Antibodies to CTLA-4 when co-administered with suboptimal concentrations of anti-CD28 Mab, enhance T cell proliferation.
• antibodies to CTLA-4 when cross-linked, antibodies to CTLA-4 profoundly inhibit the proliferation of naive T cells.
• CTLA-4 cannot replace CD28 to provide the costimulatory signal in CD28-deficient mice.
• CTLA-4 binding to its ligand(s) removes inhibitory signals, whereas aggregation (i.e. cross-linking) of CTLA-4 induces inhibitory signals which down-regulate T cell responses.
• This hypothesis was confirmed by the phenotype of CTLA-4 deficient mice which exhibit a lymphadenopathy of extreme magnitude (Waterhouse et al., 1995). The peripheral organs of these mice contain 5-10 times the normal number of lymphocytes, the vast majority of which are activated T cells as indicated by the expression of various activation markers. The latter findings clearly support the hypothesis that the function of CTLA-4 is to switch-off T cell responses which has, as we will discuss below, important implications for the design of optimal immunosuppressive strategies based on blocking the B7-CD28 interaction.
• CTIA-4Ig 50 ⁇ g/d
• IV intravenously
• Blockage of the CD28-pathway wih CTLA-4Ig (500 ⁇ g/d TV or IP on days 0, 1,2,3,4,6, and 8) resulted in significant prolongation of small bowel transplant survival in rats compared to controls, although all grafts were rejected after
• CTLA-4 Ig 100 or 250 ⁇ g/d IP for 4 weeks
• treatment with CTLA-4 Ig could reduce lethal murine GVHD in recipients of fully allogeneic bone marrow and significantly prolonged survival rates with up to 63% of mice surviving greater than 3 months post-transplantation (Blazar et al., 1994).
• the failure of CTLA-4Ig alone to induce anergy m vitro and m vivo, can most likely be explained by persistent
• IL-2 production induced by TCR triggering in combination with signaling from other accessory molecules on APC.
• GVHD graft-versus-host disease
• Acute GVHD involves necrosis of the epithelium of the skm, liver and gastrointestinal tract and is clinically characterized by skin rash, jaundice and diarrhea
• Chronic GVHD involves fibrosis and atrophy of the same organs as for acute GVHD and may result rn complete dysfunction of these affected organs
• Treatment -with both CsA and methotrexate have been shown to reduce the risk of acute GVHD
• Treatment with prednisone, alone or in combination with azathioprrne has been shown to effectively reverse chrome GVHD in 50-75% of patients.
• B7.1/B7.2-CD28 interaction could block auto-antibody production and prolongation of life in a murine model of autoimmune disease that closely resembles systemic lupus erythematosus in humans (Finck et al., 1994).
• B7.1 and B7.2 administration of a molecule which cross-links, or cross-reacts with, B7.1 and B7.2 and which does not comprise the extracellular domain of CTLA4 or CD28, can, possibly in combination with a reduced amount of immunosuppressive agents, more efficiently prevent autoimmune disorders.
• IgE production by B cells. This IgE will first sensitize local mast cells, and 'spill-over' IgE enters the circulation and binds on receptors on circulating basophils and tissue mast cells throughout the body. The IgE production is controlled by T helper 2 (Th2) cells, as IL-4 promotes the IgE synthesis by B cells.
• Th2 T helper 2
• CTLA4-Ig alone fails to induce anergy
• LFA 1 CD 1 la/CD 18
• LFA-3 CD58
• ICAlvl's CD54 CD102 CD50
• signalling through the IL-2 receptor gamma chain shortly after TCR triggering is able to prevent induction of anergy (Boussiotis et al 1994 Van Gool et al 1994)
• This pnenomenon was illustrated m an experiment in which purified T cells were cultured for six days with ⁇ llo ⁇ ntigen-expressing EBV-tr ⁇ nsformed B cells.
• the present invention aims at providing a molecule which cross-links, or cross-reacts with, B7.1 and B7.2, and which does not comprise the extracellular domain of CTLA-4 or CD28.
• CTLA4-Ig is a high affinity ligand for both B7 molecules, however this high affinity is due to a very high on-rate.
• CTLA4-Ig has also an extremely fast off-rate (Linsley et al., 1995). Since both B7 molecules are not modulated from the cell surface after ligation with CTLA4-Ig or with specific mAb's, a molecule with a very slow off-rate is the ideal blocking agent for application in vivo.
• CTLA4-Ig counter receptors There are at least 3 CTLA4-Ig counter receptors. If any of these CTLA4-Ig counter receptors can interact with CTLA-4 but not with CD28 then the usage of CTLA4-Ig may mediate deleterious effects m vivo Because CTLA4 on activated T cells functions as a terminator of T cell activation one would prefer not to block a ligand that can interact with CTLA4 but not with CD28 Such a CTLA4 ligand was demonstrated recently (Gribben et al 1995) This as yet uncharacterised CTLA4 ligand induced antigen- specific apoptosis of previously activated T cells Neither B7 1 nor B7 2 mediate apoptosis via CTLA4 Activated T cells were rechallenged with an alloantigen-bearing EBV -positive B cell line m the presence of blocking agents The simultaneous addition of mAb's to B7 1 and B7 2 but not each of them separately strongly inhibited T cell proliferation
• the present invention cams at providing in a less-complex, more-ethically acceptable and more elegant manner a molecule which cross-links or cross-reacts with B7 1 and B7 2 and which does not comprise a variable domain of a monkey antibody
• Zhang and Johnson (1997) have also snown that macaque B7 1 and B7 2 molecules are highly homologues (an overall am o acid homology of greater that 90%) to their human counterparts and are specifically recognized by neutralizing murine anti-human B7 1 and B7 2 monoclonal antibodies
• These irndings strongly suggest that macaques will develop an auto-immune response upon mjecting/immunizmg them with human B7 1 and B7 2 as described m WO 96/40878 to
• the present invention aims at providing a molecule which cross-links, or cross-reacts with, B7.1 and B7.2, as described above and which comprises at least one first domain which binds B7.1 or B7.2 or cross-reacts with B7.1 and B7.2, at least one second domain which binds B7.1 or B7.2 or cross-reacts with B7.1 and B7.2, and optionally a third domain which couples the first and the second domcdn(s).
• the present invention aims at providing a molecule as described above wherein said first and second domain is a low-molecular weight nonpeptide molecule or a polypeptide such as an antibody or a humanized antibody, a single chain fragment or another fragment thereof which has largely retained the specificity of scdd antibody, or a small antigen-binding peptide which is neither an antibody nor derived from an antibody, and wherein said third domain is a polypeptide, any chemical coupling agent or any oligomerization domain.
• the present invention aims at providing bispecific antibodies such as miniantibodies, diabodies and bispecific tetravalent antibodies, trivalent antibodies, bispecific small antigen-binding peptides and bispecific low molecular weight nonpeptide molecules.
• the invention aims at providing a method to produce said molecule which cross-links, or cross-reacts with, B7.1 and B7.2, as described above.
• the invention also aims at providing a composition comprising said molecule which cross-links, or cross-reacts with, B7.1 and B7.2, and which does not comprise the extracellular domain of CTLA-4 or CD28, or a variable domain of a monkey antibody, in a pharmaceutically acceptable excipient which can be used as a medicament.
• the invention also aims at providing an alternative and better method to inhibit antigen-specific T cell activation and/or to treat diseases of the immune system such as allograft rejection, GVHD, allergy and autoimmune diseases by using a molecule which cross-links, or cross-reacts with, B7.1 and B7.2 as described above.
• diseases of the immune system such as allograft rejection, GVHD, allergy and autoimmune diseases
• the present inventors have been able to prove that, surprisingly, cross-linking of B7.1 and B7.2 using a molecule as described above leads to the inhibition of immunoactivators such as IL- 12. This characteristic is a valuable asset in order to suppress T cell mediated immune responses.
• the present invention aims at providing a molecule which cross-links, or cross-reacts with, B7 1 and B7 2, and which does not comprise the extracellular domain of CTLA-4 or CD28 or a variable domain of a monkey antibody which possibly can be administered in conjunction with CsA or other lmmunosuppressive chemicals in order to inhibit antigen-specific T cell activation and/or to treat diseases of the immune system such as allograft rejection, GVHD, allergy and autoimmune
• Figure 1 shows the Map of baculo transfer vector pVL-Fc The vector contains
• FIG. 2 shows the protein sequence of the hB7.
• lFc soluble fusion protein Amino Acids 1- 34 : potential eukaryotic secretory signal peptide
• Amino Acids 35-241 extracellular domain of hB7.1 protein (mature protein)
• Amino Acids 242- 248 introduced by PCR cloning strategy -Amino Acids 249-480 : human IGgl-Fc (Hinge-CH2-CH3)
• Figure 3 shows the protein sequence of the hB7.2Fc soluble fusion protein Amino Acids 1-16 potential eukaryotic secretory signal sequence (Azuma et al., Nature, 1993) Amino Acids 17-239 : extracellular domain of hB7.2 protein (mature protein)
• Amino Acids 240-245 introduced by PCR cloning strategy
• Amino Acids 246-477 human IGgl-Fc (Hinge-CH2-CH3)
• Figure 4 shows the protein sequence of the hB7.1glu-glu soluble fusion protein Amino Acids 1-34 : potential eukaryotic secretory signal peptide
• Amino Acids 35-242 -extracellular domain of hB7.1 protein (mature protein)
• Amino Acids 243-251 glu-glu detection/purification tag
• Figure 5 shows the protein sequence of the hB7.2his soluble fusion protein Amino Acids 1-23 : potential eukaryotic secretory signal peptide (Azuma et al., Nature, 1993)
• Amino Acids 24-238 extracellular domain of hB7.2 protein (mature protein)
• Amino Acids 239-244 histidine detection/purification tag
• Figure 8 shows the neutralizing activity of scFv B7-24compared to parent B7-24 mAb
• Figure 9 shows the Ph ⁇ gemidpCES 1 antibody genes V L -C L variable (V) and constant (C) region of the light chain V H -C H1 , variable and first constant region of the heavy chain PlacZ promoter rbs, nbosome binding site, S signal sequence H6 six histidines stretch for IMAC purification, tag, c-myc-de ⁇ ved tag amber amber codon that allows production of soluble Fab fragments in non-suppressor strains, gill, gene encoding one of the minor coat protems of filamentous phage Restriction sites used for cloning are indicated
• Figure 10 shows sequences of hybrid immunoglobulin light chcan/CTLA-4 molecule
• Figure 1 1 shows the experimental strategy for 'random DNA shuffling'
• Figure 12 shows sequences of the CTLA-4CDR3/Vk light chain spiking oligonucleotides
• Figure 13 shows sequences of CTLA-4 CDR3/ V ⁇ light chain spiking oligonucleotides
• Figure 14 shows a BiTAb molecule
• Figure 15 shows the DNA sequence of the B ⁇ TAbB7-24- 1 G 10H6 molecule
• Nucleotides 1-72 pelB signal sequence Nucleotides 73- 415 VH region anti B7 1 Mab (B7-24) Nucleotides 416 - 460 (G 4 S) 3 flexible linker Nucleotides 461 -787 VL region anti B7 1 Mab (B7-24) Nucleotides 788 - 820 Human IgG3 Lunge region
• Nucleotides 821 - 925 Helix-Turn Helix Dime ⁇ sation Domain Nucleotides 926 958 Human IgG3 Lunge domain Nucleotides 959 1325 VH region anti B7 2 Mab ( 1G10) Nucleotides 1326 - 1369 (G 4 S0 flexible linker Nucleotides 1370- 1708 VL region anti B7 2 Mab ( 1 G 10)
• Nucleotides 1709 - 1726 H ⁇ s6 Tag Figure 16 shows the Protein Sequence of the BiTAbB7-24-lG10H6 molecule Amino Acids 1-24: pelB signal sequence Amino Acids 25- 138: VH region anti B7.1 Mab Amino Acids 139- 153: (G 4 S) 3 flexible linker Amino Acids 154 - 262: VL region anti B7.1 Mab
• Amino Acids 263- 273 Human IgG3 hinge region
• Amino Acids 274-308 Helix-Turn-Helix Dimerisation Domain
• Amino Acids 309-319 Human IgG3 hinge domain
• Amino Acids 320-446 VH region anti B7.2 Mab
• Amino Acids 447- 461 (G 4 S) 3 flexible linker
• Amino Acids 462- 574 VL region anti B7.2 Mab Amino Acids 575- 580: His6 Tag
• Figure 17 shows DNA sequence the BiTAblG10-B7-24H6 molecule Nucleotides 1 - 360 : VH region anti B7.2 Mab ( 1G10)
• Nucleotides 361 - 405 (G 4 S) 3 flexible linker Nucleotides 406 - 744 : VL region anti B7.2 Mab (1G10) Nucleotides 745 - 777 : Human IgG3 hinge region Nucleotides 778 - 882 : Helix-Turn-Helix Dimerisation Domain Nucleotides 883 - 915 : Human IgG3 hinge domain
• Nucleotides 916 - 1278 VH region anti B7.1 Mab (B7-24)
• Nucleotides 1279 - 1314 (G 4 S) 3 flexible linker
• Nucleotides 1314 - 1650 VL region anti B7.1 Mab (B7-24)
• Nucleotides 1651 - 1670 His6 Tag
• Figure 18 shows Protem Sequence of the BiTAb lG10-B7-24H6 molecule Amino Acids 1 - 120 : VH region anti B7.2 Mab Amino Acids 120 - 135 : (G 4 S), flexible linker Amino Acids 136 - 248 : VL region anti B7.2 Mab Amino Acids 249 - 259 : Human IgG3 hinge region
• Amino Acids 260 - 285 Helix-Turn-Helix Dimerisation Domain Amino Acids 286 - 305 : Human IgG3 hinge domain Amino Acids 306 - 426 : VH region anti B7.1 Mab Amino Acids 427 - 441 : (G 4 S) 3 flexible linker Amino Acids 442 - 550 : VL region anti B7.1 Mab Amino Acids 551 - 556 : His6 Tag
• Figure 19 show ⁇ s the DNA sequence of the dimerisation domain HDH Nucleotides 1 - 33 : Human IgG3 hinge region Nucleotides 34 - 82 : helix-domcrin Nucleotides 83 - 90 : turn
• Nucleotides 140 - 171 Human IgG3 hinge region
• Figure 20 shows the Protein sequence of the dimerisation domain HDH Amino Acids 1 - 1 1 : Human IgG3 hinge region
• Figure 21 shows the DNA sequence of the dimerisation domain JEM- 1
• Figure 22 shows the Protein se ⁇ uence of the dimerisation domain JEM-1 Amino Acids 1- 33 : JEM-1 dimerisation domain
• Figure 23 shows DNA sequence of monospecific Diabody B7-24: VH-B7-24/5/VL-B7- 24/H6
• Nucleotides 1-72 pelB signal sequence
• Nucleotides 73 - 414 VH region anti B7.1 Mab (B7-24)
• Nucleotides 415 -429 G 4 S flexible linker
• Nucleotides 430 - 756 VL region ⁇ nti B7.1 Mob (B7-24)
• Nucleotides 757 - 773 His6 Tag
• Figure 24 shows Protein Sequence of monospecific Diabody B7-24: VH-B7-24/5/VL- B7-24/H6
• Amino Acids 1- 24 pelB signal sequence
• Amino Acids 25 - 138 VH region anti B7.1 Mab
• Amino Acids 139 - 143 G 4 S flexible linker
• Amino Acids 144 - 252 VL region anti B7.1 Mab
• Amino Acids 253 - 259 His6 Tag
• Figure 25 shows DNA sequence of monospecific Diabody 1G10: VH-1G10/5/VL- 1G10/H6
• Nucleotides 1-72 pelB signal se ⁇ uence
• Nucleotides 73 - 433 VH region anti B7.2 Mab (1G10)
• Nucleotides 434- 447 G 4 S flexible linker Nucleotides 448 - 786 : VL region anti B7.2 Mab (1G10) Nucleotides 787 - 804 : His6 Tag
• Figure 26 shows Protein Se ⁇ uence of monospecific Diabody 1G10: VH- 1 G 10/5/VL-
• Amino Acids 1 - 24 pelB signal sequence
• Amino Acids 25 -144 VH region anti B7.2 Mab
• Amino Acids 145 - 149 G 4 S flexible linker
• Amino Acids 150 - 262 VL region anti B7.2 Mab
• Figure 27 shows DNA se ⁇ uence of bispecific Diabody I : VH-1G 10/5 VL-B7-24/H6 Nucleotides 1-1 17 : g3p - signal sequence Nucleotides 1 18 - 483 : VH region anti B7.2 Mab ( 1G10)
• Nucleotides 484 - 498 G,S flexible linker Nucleotides 499 - 825 VL region ⁇ nti B7 1 Mob (B7 24) Nucleotides 826 - 843 H ⁇ s6 Tag
• Figure 28 shows the Protem sequence of bispecific Diabody I VH 1 G 10/5/VL-B7- 24/H6
• Figure 29 shows the DNA sequence of bispecific Diabody II VH B7-24/5/VL IGl 0 Nucleotides 1 1 17 g3p signal se ⁇ uence Nucleotides 1 18 - 465 VH region anti B7 1 Mab (B7-24) Nucleotides 466 - 480 G 4 S flexible linker
• Figure 30 shows the Protem se ⁇ uence of bispecific Diabody II VH-B7-24/5/VL-1G10 Amino Acids 1 - 39 g3p - signal sequence Amnno Acids 40 - 155 VH region anti B7 1 Mab
• Figure 31 shows tne DNA se ⁇ uence of monospecific T ⁇ abody B7 24 VH-B7- 24/0/VL B7-24H6
• Nucleotides 1-72 pelB signal se ⁇ uence Nucleotides 73 - 414 VH region anti B7 1 Mab (B7-24) Nucleotides 415 - 741 VL region anti B7 1 Mab (B7 24) Nucleotides 742 - 759 H ⁇ s6 Tag
• Figure 32 shows the Protein sequence of monospecific T ⁇ abody B7-24 VH-B7 24/0NL-B7-24H6
• Amino Acids 1- 24 pelB signal se ⁇ uence Amino Acids 25 - 138 : VH region anti B7.1 Mab Amino Acids 139 - 247 : VL region anti B7.1 Mab Amino Acids 248 - 253 : His6 Tag
• Figure 33 shows the DNA sequence of monospecific Triabody 1G10 : VH-1G10/0/VL- 1G10H6
• Nucleotides 1-72 pelB signal sequence
• Nucleotides 72 - 433 VH region anti B7.2 Mab (1G10)
• Nucleotides 434 - 771 VL region anti B7.2 Mab ( 1G10) Nucleotides 772 - 789 : His6 Tag
• Figure 34 shows the Protein sequence of monospecific Triabody 1G10 : VH- 1G10/0/VL-1G10H6
• Figure 35 shows the DNA sequence of bispecific Triabody I : VH- 1 G 10/0/VL-B7-
• Nucleotides 1 18 - 483 VH region anti B7.2 Mab (1G10)
• Nucleotides 484 - 810 VL region anti B7.1 Mab (B7-24)
• Figure 36 shows the Protein sequence of bispecific Triabody I : VH- 1G10/0/VL-B7- 24/H6 Amino Acids 1- 39 : g3p - signal se ⁇ uence
• Amino Acids 40 - 161 VH region anti B7.2 Mab Ammo Acids 162 - 270 VL region ⁇ nti B7 1 Mob Ammo Acids 271 - 276 H ⁇ s6 Tag
• Figure 37 shows the DNA sequence of bispecific Triabody II VH B7-24/0A ⁇ L-lG10 Nucleotides 1-1 17 g3p - signal sequence
• Figure 38 shows the Protem sequence of bispec ⁇ icT ⁇ abody II VH-B7-24/0/VL- 1 G 10 Ammo Acids 1- 39 g3p - signal sequence
• Figure 39 shows the geliiltration profile of scFv B7-24 LO (a) scFV B7-24 L5 (b) and scFv B7-24 L15 (c)
• Figure 40 shows the bmdmg of unpurified scFv B7-24, B7-24 diabodies (scFv B7-24 L5)- and B7-24 tnabodies (scFV B7-24 L0) on B7 1 ED fusion protein
• Figure 41 shows the bmdmg of unpurified scFv 1 G 10 1 G 10 diabodies (scFv 1 G 10
• Figure 42 shows bmdmg of unpurified scFv B7-24 B7-24 diabodies (scFv B7-24 L5)- and B7-24 tnabodies (scFV B7-24 L0) on different 3T6 cells
• Figure 43 shows binding of semi-pu ⁇ fied scFv B7 24 B7 24 diabodies (scFv B7 24 L5)- and B7-24 tnabodies (scFV B7-24 L0) on B7 1 ED fusion protein
• Figure 44 shows Joindrng of semi-purified scFv B7-24 B7-24 diabodies (scF 1 " B7-24 L5)- and B7-24 tnabodies (scFV B7-24 L0) on RPMI8866 cells
• Figure 45 shows the neutralizing activity of scFv B7-24 and B7-24 diabodies (scFv
• Figure 46 shows the neutralizing activity of scFv B7-24 and B7-24 tnabodies (scFV B7-24 LO) a MLR
• Figure 47 shows bmdmg of B7-24 diabodies (scFv B7-24 L5)- and B7-24 tnabodies (scFV B7-24 LO) gelfiltration fractions on B7 1ED fusion protem
• Figure 48 shows the gelfiltration of B7-24/1G 10 crosslinked monoclonal antibodies
• Figure 49 shows the bmdmg of the crosshnked monoclonal antibodies on 3T6 cells
• Figure 50 shows the Biacore results of the crosshnked b7-24/lG10 monoclonal antibodies
• the present invention is based on the finding that molecules which crosslink, or cross-react with, B7 1 and B7 2, and which do not comprise a variable domain of a monkey antibody or the extracellular domain of CTLA-4 or CD28 efficiently inhibit antigen specific T cell activation Accordingly these molecules can be used to prevent or treat (terms used interchangeably) diseases such as transplant rejection GVHD allergy and autoimmune diseases where antigen- specific T cell activation is pathogenic More particularb the present invention relates to a molecule which crosslinks or cross reacts with B7 1 and B7 2 and which does not comprise a variable domain of a monkey antibody or the extracellular domain of CTLA-4 or CD28 resulting in the inhibition of antigen-specific T cell activation
• molecule which cross-reacts with B7 1 and B7 2 and which does not comprise the extracellular domain of CTLA-4 or CD28 refers to any molecule known m the art which simultaneously binds (not necessarily to a common epitope of B 1 and B7 2) or binds to a common epitope of B7 1 (dec ⁇ bed by Freeman et al , 1989) and B7 2 (described by Freeman et al , 1993 and Azuma et al , 1993) and which does not comprise the extracellular domain of CTLA-4 or CD28 Molecules compnsmg the extracellular domains of CTLA-4 or CD28, such as CTLA-4Ig or CD28Ig respectively, are known to be able to cross-link, or crossreact with B7 1 and B7 2 (described m US patent N c 5 434, 131 and 5 521,288 to linsley et al respectively)
• CTLA-4Ig the extracellular domains of CTLA-4 or CD28
• variable domain of a monkey antibody specifically refer to the variable domains of the macaque monoclonal antibodies 7B6, 16C10, 7C10 and 20C9 described in WO 96/40878 (PCT/US96/10053) to Anderson et al. The latter reference is incorporated by reference in its entirety herein.
• the invention relates to a molecule which comprises at least one first domain which binds B7.1 or B7.2 or cross-reacts with B7.1 and B7.2, at least one second domain which binds B7.1 or B7.2 or cross-reacts with B7.1 and B7.2, and optionally a third domain which couples the first and the second domcrin(s).
• domain refers to any antibody-like polypeptide, any nonantibody-like peptide or any nonpeptide organic molecule.
• B7.12 diabodies which bind in a monovalent fashion to B7.1 and B7.2 or which bind in a bivalent fashion to a common epitope of B7.1 and B7.2
• B7.12 triabodies which bind simultaneously in a monovalent fashion to B7.1 and bivalently to B7.2 or in a monovalent fashion to B7.2 and bivalently to B7.1 or trivalently to B7.1
• tetravalent antibodies which bind bivalently to B7.1 and B7.2 or tetravalently to B7.1
• small antigen binding peptides or low molecular weight nonpeptide molecules which bind mono-or multivalently to B7.1, B7.2 and/or B7.12.
• any chemical couphng agent or any oligomerization domain refers to any molecule known in the art which is capable of coupling the said first and second domains to each other.
• Examples of such domains are the known leucine zipper of c-fos and c-jun (Kostelny et al, 1992; de
• the term 'antibody' refers to polyclonal or monoclonal antibodies
• the term “monoclonal antibody' refers to an antibody composition having a homogeneous antibody population The term is not hmited regarding the species or source of the antibody nor is it intended to be limited by the manner m which it is made
• the term "humanized antibody” means that at least a portion of the framework regions of an lmmunoglobuhn are derived from human immunoglobulin sequences
• the term 'single chain antibody refers to antibodies prepared by determining the bmdmg domains (both heavy and tight chains) of a bmdmg antiboav and supplymg a hnkmg moiety which permits preservation of the binding iunction This forms in essence a radically abbreviated antibody having only that part of the variable domain necessary for bmdmg the antigen Determination and construction of single chain antibodies are described in U S
• fragments refers to F ab F (ab)2 , F and other fragments which retain the antigen bmdmg function and specificity of the parent antibody
• Antibodies to human B7 1 and human B7 2 are known in the art
• the present invention contemplates a new use lor such antibodies as detailed above
• Monoclonal antibody B7 24 was prepared as described m the international application WO 94/01547
• Monoclonal antibodies 5B5 and 1G10 were prepared essentially as described m U S Patent No 5 397 703 or international application WO 94/01547 and can be obtained at Innogenetic ⁇ N V Indust ⁇ epark Zv jnaarde 7 box 4 B
• Monoclonal antibodies 3H 10 5F3 7B8 9D8 1 1B9 13B9 13D3 and Fl were prepared as described in example 1 (see further) and can be obtained at Innogene ⁇ cs N V Indust ⁇ epark Zwijnaarde 7 box 4 B 9052 Ghent Belgium fax
• the present invention more specifically relates to miniantibodies, diabodies, triabodies, tetravalent antibodies, antigen-binding peptides and low molecular weight nonpeptide molecules which cross-link, or cross-react with, B7.1 and B7.2, and do not comprise a variable domain of a monkey antibody or the extracellular domain of CTLA-4 or CD28, and which are produced by the following methods:
• hybridomas derived from immunized transgenic mice, containing large sections of the human immunoglobulin (Ig) gene loci in the germ line, integrated by the yeast artificial chromosomal (YAC) technology, resulting in effective blocking antibodies as described by Mendez et al ( 1997).
• small antigen-binding peptides or fragments refers to any peptide (i.e. a polymer composed of at least two amino acids) which cross-links, or cross-reacts with, B7.1 and B7.2, and which does not comprise a variable domain of a monkey antibody or the extracellular domain of CTLA-4 or CD28.
• low-molecular weight nonpeptide molecules refers to any molecule which is not a peptide and which cross-links, or cross-reacts with, B7.1 and B7.2, and which does not comprise a variable domain of a monkey antibody or the extracellular domain of CTLA-4 or CD28.
• the present invention further relates to a composition
• a composition comprising a molecule which cross-links, or cross-reacts with, B7.1 and B7.2, and which does not comprise a variable domain of a monkey antibody or the extracellular domain of CTLA-4 or CD28 in a pharmaceutically acceptable excipient, possibly in combination with immunosuppressive agents such as cyclosporin A (Sandimmune, Neoral; Sandoz-Sangstat), FK 506 (Tacrolimus, Prograf; Fujisawa), rapamycin (Sirolimus; American Home Products), OKT-3 (anti-CD3 mAb; Johnson & Johnson),
• immunosuppressive agents such as cyclosporin A (Sandimmune, Neoral; Sandoz-Sangstat), FK 506 (Tacrolimus, Prograf; Fujisawa), rapamycin (Sirolimus; American Home Products), OKT-3 (anti-CD3 mAb; Johnson & Johnson),
• OKT-4 anti-CD4 mAb;Johnson & Johnson
• SB-210396 anti-CD4 mAb; Smimkline Beecham
• T10B9 anti-TcR antibody; Medlmmune
• BTI 322 anti-CD2 mAb; Biotransplant
• Mycophenolate mofetil Cellcept; Roche
• anti-thymocyte immunoglobulin Thymoglobulin (rabbit); Pasteur Merieux), anti-lymphocyte immunoglobulin (Lymphoglobulin (equine); Pasteur Merieux), anti-lymphocyte immunoglobulin (ATG Fresenius (rabbit); Hoechst Marion Roussel), azathioprine (Imuran; Glaxo Wellcome), leflunomide (Hoechst Marion Roussel), triple therapy combining cyclosporin A (Sandimmune, Neoral; Sandoz-Sangstat) with azathioprine (Imuran; Glaxo Wellcome) and glucocorticoster
• composition refers to any composition compnsmg as an active mgredient a molecule which cross-hnks, or cross-reacts with B7 1 and
• B7 2 and which does not comprise a variable domain of a monkey antibody or the extracellular domain of CTLA-4 or CD28 according to the present invention possibly m the presence of suitable excipients known to the skilled man
• CTLA-4 or CD28 of the invention may thus be administered m the form of any suitable composition as detailed below by any suitable method of administration within the knowledge of a skilled man
• the preferred route of administration is parenterally
• the compositions of this invention will be formulated in a unit dosage rnjectable form such as a solution suspension or emulsion m association with a pharmaceutically acceptable excipient
• excipients are inherently nontoxic and nontherapeutic Examples of such excipients are saline, Ringer's solution, dextrose solution and Hank's solution
• Nonaqueous excipients such as fixed oils and ethyl oleate may also be used
• a preferred excipient is 5% dextrose rn saline
• the excipient may contain minor amounts of additives such as substances that enhance lsotomcity and chemical stability including buffers and preservatives
• the molecule which cross-hnks, or cross-reacts with, B7 1 and B7 2 and which does not comprise a variable domain of a monkey anubody or the extracellular domain of CTLA-4 or CD28 of the invention are administered at a concentration that is therapeutically effective to prevent allograft rejection GVHD allergy and autoimmune diseases
• the dosage and mode of administration will depend on the individual Generally the compositions are administered so that the molecule cross-lmks or cross-reacts with B7 1 and B7 2 and which does not comprise a variable domain of a monkey antibody or the extracellular domain of
• CTLA-4 or CD28 is given at a aose between 1 ⁇ g/kg and 10 mg/kg more preferably between 10 ⁇ g/kg and 5 mg/kg most preferably between 0 1 and 2 mg/kg
• it is given as a bolus dose Contmuous short time infusion (durmg 30 minutes) may also be used
• the molecule which cross-links or cross-reacts with, B7 1 and B7 2 and which does not comprise a variable domain of a monkey antibody or the extracellular domain of CTLA-4 or CD28 constructs or compositions compnsmg the same may be infused at a dose between 5 and 20 ⁇ g/kg/minute, more preferably between 7 and 15 ⁇ g/kg/mmute
• CTLA-4 or CD28 needed should be determined as bemg the amount sufficient to cure the patient in need of treatment or at least to partially arrest the disease and its complications Amounts effective for such use will depend on the seventy of the disease and the general state of the patient's health Single or multiple administrations may be required depending on the dosage and frequency as required and tolerated by the patient
• molecules of the present invention or the compositions compnsmg the same may be administerea before during or after the organ transplantation as is desired from case to case
• the molecules or the compositions comprising the same are administered directly to the host treatment will preferably start at the time of the transplantation and continue afterwards m order to prevent the activation and differentiation of host T cells against the MHC on the allograft
• the donor organ is ex vivo pe ⁇ used with molecules or the compositions comprising the same treatment of the donor organ ex vivo will start before the time of the transplantation of the donor organ m order to prevent the activation and differentiation of host T cells agarnst the MHC on the allograft
• mice One female BALB/c mice was immunized (injected intraperitonec ly) four times (i.e., at days 0, 13, 26 and 213) with 5x10 C Sf9 insect cells that were infected with a recombinant baculovirus containing a human B7.1 cDNA.
• spleen cells were retrieved from the immunized mice and used for cell fusion mainly according the procedure as described by K ⁇ hler and Milstein ( 1975).
• dissociated splenocytes from the immunized mice were fused with murine myeloma cells SP2/0-Agl4 (ATCC, CRL-1581) at a ratio of 10:3 using a polyethylene glycol/DMSO solution.
• the fused cells were mixed up and resuspended in DMEM medium supplemented with hypoxanthine, thymidine, sodium pyruvate, glutamine, a non-essential amino acid solution, 10% inactivated fetal calf serum, 10% inactivated horse serum and 10% BM-Condimed.
• the cells were then distributed to 960 wells on tissue culture plates to which aminopterin was added 24 hours after the cell fusion. Each well contained between 1 to 5 growing hybridoma clones at the average. After eight days supernatants of the 960 wells were combined in groups of 10 for primary screening. The 96 pools were screened for the presence of specific antibody by FACS analysis using the B7.1 positive
• Epstein-Barr virus ( ⁇ BV)-transformed human B cell fine, ARC Epstein-Barr virus ( ⁇ BV)-transformed human B cell fine, ARC .
• cells (5xl0 5 cells/sample) were incubated for 30' at 4 ⁇ C with the different (100 ml undiluted) supernatants. .
• FTTC fluoresceine isothiocyanate
• This second screening provided one individual positive well containing antibodies reactive with the B7.1- expressing ARC cells This positive well was subcloned and one stable hybridoma clone named 5B5 was obtained. This hybridoma clone secrete mouse antibodies of the IgG3 isotype.
• the antibody secreted by the hybridoma clone 5B5 was tested for specific binding to the human B7.1 molecule in a FACS experiment using the B7.1 -expressing ARC cells or the mouse fibroblasts, 3T6 cells, transfected with cDNA encoding human B7.1 molecule (3T6-B7.1 cellsXDe boer et al., 1992). As control non-transfected 3T6 cells were used. Cells (2.5x10 5 cells/sample) were incubated with the supernatant of the hybridoma clone 5B5 for 30 min. at 4°C.
• the cells were washed (PBS supplemented with 2% inactivated FCS ands 0.02% azide) three times and incubated with FTTC-labeled goat anti-mouse antiserum (i.e. GAM-FTTC). The cells were also incubated with the GAM-FTTC alone. After another 3 washes, the cells were analysed for fluorescent staining using a FACScan instument. Results showed that the antibodies secreted by the 5B5 hybridoma clones specifically bound to the B7.1 -expressing ARC and 3T6/B7.1 cells whereas 3T6 cells that do not express B7.1, did not exhibit any binding significantly greater than that of the control monoclonal antibody or GAM-FTTC.
• the antibodies, secreted by the 5B5 hybridoma clone were tested for their capacity to inhibit the proliferation of human peripheral blood T lymphocytes, activated with anti-CD3 (OKT-3) in the presence of the mouse 3T6 cells transfected with the cDNA encoding human B7.1 molecule and human CD32 (3T6/CD32/B7.1 )
• T lymphocytes Human peripheral blood T lymphocytes were isolated from buffy coat by density centriiugation.T cells were further purified by cold aggregation of the monocytes (4X 10 6 PBMC/ml in RPMI bic + 10 % iFCS, 30' rotation at 4 °C; 15' on ice to separate the monocyte aggregates; supernatant contains the enriched T cells). T cells were further enriched by depletion of monocytes, B cells and NK cells using Lympho-Kwik T (One Lambda, Los Angeles, CA) according to the manufacturers protocol.
• Lympho-Kwik T One Lambda, Los Angeles, CA
• mice Two female BALB/c mice were immunized (injected intraperitoneally) four times (i.e., at days 0, 28, 56 and 208) with Sf9 insect cells that were infected with a recombinant baculovirus containing a human B7.2 cDNA. Three days after the last injection, spleen cells were retrieved from the immunized mice and used for cell fusion mainly according to the procedure as described by Kohler and Milstein ( 1975). Dissociated splenocytes from the immunized mice were fused with SP2/0 murine myeloma cells at a ratio of 10:3 using a polyethylene glycol/DMSO solution. The fused cells, derived from the two mice, were mixed up and resuspended in
• DMEM medium supplemented with hypoxanthine, thymidine, sodium pyruvate, glutamine, a non-essential amino acid solution, 10% inactivated fetal calf serum and 10% inactivated horse serum.
• the cells were then distributed to 1440 wells on tissue culture plates to which arriinopterin was added 24 hours after the cell fusion. Each well contained between 1 to 5 growing hybridoma clones at the average.
• the cells were washed twice in RPMI 1640 supplemented with 10% FCS and finally suspended in PBS supplemented with 1% BSA and 0.1 % NaN 3 and analyzed with a FACScan flow- cytometer (Becton Dickinson).
• the specific binding of the monoclonal antibodies is expressed as the mean fluorescent intensity in arbitrary units.
• This first screening yielded eighteen positive pools.
• the ninety wells corresponding to the eighteen positive pools were subjected to a second screening, with the FACS screening technique described above using the B7.2-expressing human EBV-transformed B cell line RPMI 6688.
• This second screening provided eleven individual positive wells containing antibodies reactive with the 37.2-expressing EBV-transformed human B cell fine RPMI 8866. These positive wells were subcloned and eight stable hybridoma clones named 3H10, 5F3, 7B8, 9D8, 1 1B9, 13B9, 13D3, 14F1 were obtained. These hybridoma clones secrete mouse antibodies of different IgG isotypes: IgGl :9D8;
• IgG2a 5F3, 7B8, 14F1; IgG2b: 3H10, 1 1B9, 13B9, 13D3.
• the antibodies secreted by hybridoma clones 3H 10 5F3, 7B8 9D8 1 1B9 13B9, 13D3, 14F1 were tested for specific bmdmg to the human B7 2 molecule B7 2- expressing EBV-transformed human B cells (RPMI 8866), freshly isolated peripheral blood human T cells and monocytes were incubated with the supernatant of the different hybridoma clones 3H 10, 5F3, 7B8, 9D8 11B9, 13B9 13D3 14Fl or an isotype matched control monoclonal antibody (control mAb) for 30 mm at 4°C Thereafter, the cehs were separated from the supernatant, washed three times and incubated with FTTC-labeled goat anti-mouse IgG antiserum d e GAM-FTTC)
• the cehs were pulsed for 6 to 8 h with 1 ⁇ Ci [ 3 H] -Thymidine, after which the cehs were harvested usmg an automated ceh harvester [ 3 HJ- Thymidine incorporation was determined with a liquid scintillation counter.
• mice One female BALB/c mice was immunized (injected rntrapentoneally) four times d e , at days 0, 27 56 and 97) with 5x10 b Sf9 insect cehs that were infected with a recombinant baculovirus containing a human B7 2 cDNA
• 5x10 b Sf9 insect cehs that were infected with a recombinant baculovirus containing a human B7 2 cDNA
• spleen cehs were retrieved from the immunized mice and used for cell fusion mamly according the procedure as described by Kohler and Mhstem (1975) Dissociated splenocytes from the immunized mice were fused w th SP2/0 munne myeloma cehs at a ratio of 10 3 usmg a polyethylene glycol/DMSO solution
• the fused cehs were mixed up and resuspended DME medium supplemented with hypoxanthme, thymidine, sodium pyruvate glutamine a non-essential ammo acid solution 10% inactivated fetal calf serum, 10% inactivated horse serum and 10% BM-Con ⁇ med
• the cehs were then distributed to 960 wehs on tissue culture plates to which ⁇ minopte ⁇ n was added 24 hours after the cell fusion
• Each well contained between 1 to 5 growing hybridoma clones at the average
• Aiter eight days supernatants of the 960 wehs were combined in groups of 5 for primary screening.
• the 192 pools were screened for the presence of specific antibody by FACS analysis using freshly isolated monocytes. Monocytes were isolated from buffy coat on Ficoh-Paque(density gradient 1.077; Pharmacia) gradients. After three washings, monocytes were removed by cold agglutination (Mentzer et al., 1986).
• cehs were washed two times with PBS supplemented with 2% inactivated FCS ands 0.02% sodium azide and incubated with goat anti-mouse (IgG and IgM) antibodies conjugated to fluorescein isothiocyanate (FTTC) for 30' at 4°C After another two washes, cehs were fixed using 0.5% paraformaldehyde and analyzed with a FACScan flow cytometer
• the specific binding of the monoclonal antibodies is expressed as the mean fluorescent intensity in arbitrary units.
• the first screening yielded five positive pools.
• the twenty-five wehs corresponding to the five positive pools were subjected to a second screening with the FACS screening technique described above using 5xl0 5 freshly isolated monocytes for each staining.
• This second screening provided one individual positive well containing antibodies reactive with the monocytes.
• This positive weh was subcloned and the subclones were subjected to a FACS screening as described above using the B7.2 expressing EBV-transformed human B ceh hne RMPI8866, (5x10 5 cehs/stcdning).
• hybridoma clone 1G10 secrete mouse antibodies of the IgG2a isotype.
• the antibodies secreted by the hybridoma clone 1G10 were tested for specific binding to the human B7.2 molecule.
• FACS staining on mouse 3T6 cehs, tranfected with the cDNA encoding human B7.2 molecules (3T6/B7.2) and on B7.2-negative 3T6 cells was performed.
• the 3T6 and 3T6/B7.2 cells were firstly incubated for 20' at 4°C in PBS supplemented with 5% inactivated FCS and 0.02% sodium ⁇ zide and 10% normal rabbit serum Subsequently the cells were incubated with the supernatant of the hybridoma clone 1G10 for 30 mm at 4°C Thereafter the cehs were separated from the supernatant washed three times and incubated with fluorescein lsothyocyanate-labeled goat anti-mouse antiserum d e GAM-FTTC) The cehs were also incubated with the GAM-FTTC alone After another
• the antibodies secreted by the 1G10 hybridoma clone were tested for their capacity to inhibit the proliferation of human peripheral blood T lymphocytes activated with anti-CD3 (OKT-3) m the presence of the mouse 3T6 cehs transfected with the cDNA encoding human B7 2 molecule and human CD32 (3T6/CD32/B7 2)
• Human peripheral blood T lymphocytes were isolated from buffy coat by density centrifugation T cehs were further punned by cold aggregation of the monocytes (4X 10 6 PBMC/mhn RPMI bic + 10 % iFCS, 30' rotation at 4 °C 15' on ice, supernatant contains the enriched T cehs) T cehs were further enriched by depletion of monocytes, B cehs and NK cehs usmg Lympho Kwik T (One Lambda, Los Angeles CA) according to the manufacturers protocol Mitomycin C-treated
• 3T6/CD32/B7 2 cehs(at 200 ⁇ l Mitomycin C at 250 ⁇ g/ml to 800 ⁇ l RPMI bic - 10 iFCS durmg 45' at 37°C wash twice m RPMI bic ⁇ 10 % iFCS) at 10' cehs well were incubated with anti-CD3 mAb (OKT3 0 5 mg/ml) for 1 h at 37 " C fohowed by a 1 h incubation at 37 ⁇ C with decreasing concentrations of the antibodies secreted by the 1G10 hybridoma clone
• Subsequently purified T cells (5x10' cells/well) were added and incubated for 5 days After 5 aavs of culture the cehs ere pulsed for 6 to 8 h with 1 ⁇ Ci [ ⁇ ] Thymidine after which the cehs were harvested usmg an automated cell harvester 0HJ-Thymiarne incorporation was determined w th
• B7.1 or B7.2 molecules The Netherlands, which contains 4.1 x 10 10 antibody molecules was screened for binding to human B7.1 or B7.2 molecules.
• the direct selection was performed on biotinylated B7.1 ED or B7.2ED fusion proteins (B7. lED-Bio or B7.2ED-Bio) and specific phage antibodies captured by streptavidin paramagnetic particles and a magnet.
• B7.1 and B7.2 are presented as immunoglobulin fusion proteins, we performed the selection in the presence of a 10 fold molar excess of human IgG to compete out the antibodies specific for the Fc moiety. Different (4) rounds of selection were performed.
• the human Fab library was tested for binding 500 nlvl B7.1 ED-Bio
• the previous selected Fob's were tested for binding 1 OOnM B7.
• lED-Bio and in the fourth round of selection the previous selected Fob's were tested for binding 10 nM B7.1 ED-Bio.
• the selection is for antibodies specific for the native conformation of the antigen. Specific clones are screened in a phage ELISA for binding to B7.1ED or B7.2ED fusion protein.
• Ivlarval PBS Ivlarval PBS. Plates were washed a further 3 times with PBS and PBS plus tween. To each well 50ul of 4% Marval PBS was added and 50ul of phage supernatants. These were mixed by pipetting up and down and left for 1.5 hrs. Plates were washed 3 times with PBS and PBS plus tween. A secondary antibody of goat anti immunoglobulin was added to each well and incubated for 1 hour at room temp.
• Antibody was discarded and washed 3 times with both PBS and PBS tween. Positive ph ⁇ ge antibodies were then detected using standard TMB staining and detection at OD 450. The reaction was stopped by adding lOOul of 2M H 2 S0 4 . Results showed that there was a high diversity of binders to B7-lIg in the 3 rd round of selection at lOOnM. Nine different patterns were obtained . After the 4 th round of selection on B7.1-Ig (lOnM) one binder was recovered. This clone may have been selected for higher affinity by the 10 fold reduction in antigen concentration from round 3 to 4, however an increased phage display efficiency cannot be excluded as a contributing factor to the preferential selection of this clone.
• cehs were harvested by centrifugation and resuspended in ice cold shockbuffer (lOOmM Tris-HCl pH 7.4; 20% sucrose, ImM EDTA, pH8). After incubation on ice during 10' with occasional stirring, the mixture was centrifuged at lO.OOOrpm during 1,5'. The supernatans was removed and the pellet was immediately resuspended in ice cold distihed water. After incubation on ice during ice during ice cold shockbuffer (lOOmM Tris-HCl pH 7.4; 20% sucrose, ImM EDTA, pH8). After incubation on ice during 10' with occasional stirring, the mixture was centrifuged at lO.OOOrpm during 1,5'. The supernatans was removed and the pellet was immediately resuspended in ice cold distihed water. After incubation on ice during
• Soluble proteins are generated which comprise the extracellular domains (ED) of respectively human B7.1 and human B7.2 proteins carboxyterminahy fused to the Fc-domcrin of a human IgG ⁇ 1 (further referred as ED fusion protein), the peptide EEEEYMPME (glu-glu epitope) or a six histidine peptide (his) (further referred as ED-glu-glu or ED-his proteins).
• ED extracellular domains
• Ah B7 proteins are expressed in insect cehs using the recombinant baculovirus expression system (BENS). Alternative expression systems are used.
• the soluble B7.1ED and B7.2ED fusion proteins, B7.1ED glu-glu and B7.2ED his proteins are needed for the further selection and optimization of higher affinity antibodies.
• baculovmons expressmg soluble h37.1ED or h B7.2 ED fusion proteins or hB7. lglu-glu or hB7.2 his protems.
• a foreign gene expression casette consisting of the protein-encoding cD ⁇ A se ⁇ uence under transcriptional control of a strong baculoviral promoter (e.g. polyhedrin promoter), in the genome of the Autogxapha calif ornica Nuclear Polyhedrosis Virus (AcNPV), the prototype of the Baculoviridae, at a genome locus non-essential for in vitro replication of the virus (e.g. polyhedrin locus).
• baculoviral promoter e.g. polyhedrin promoter
• AcNPV Autogxapha calif ornica Nuclear Polyhedrosis Virus
• baculovirus genome As the baculovirus genome is too large, easy msertion of this recombinant expression casette (cDNA and viral promoter) by simple cloning techniques is not possible Therefore generation of the recombinant baculovirus genome was performed usmg a technology based on in vivo homologous recombination between the baculovirus w ld type genome and a transfer plasmid containing the foreign expression casette flanked by baculoviral genome sequences, both introduced msect cehs by classical cotransfection technology
• the resulting recombinant genome encodes a recombinant virus that is subsequently purified and amplified providing a high titre recombinant virus stock that can be used for large scale infection of msect cehs thereby producing large amounts of the foreign protem
• Fusion protems of both the hB7 1 and hB7.2 ED with the Fc part (H ⁇ nge-CH2-CH3) of a humanlgG ⁇ 1 immunoglobulin were expressed as recombinant baculovirus protems
• the cloning of the human IgG ⁇ 1 -Fc sequence was performed by means of RT-PCR on mRNA isolated from stimulated human B cehs ( 16h coculture with mitomycin C treated mouse fibroblast (3T6) transfected w th cDNA encoding human CD40L ⁇ gand, 3T6/hCD40L, m the presence of 20U/ml rhIL2), usmg specuic PCR-primers designed on the hlgG ⁇ 1 se ⁇ uence pubhshed by Ellison et 01.( 1982, NAR 4071)
• the PCR amplified DNA fragment consisted of a 6 ammoacid encoding linker sequence mcludmg a BamHI site fo
• fragment PVL1393 is a polyhedrin promoter controlled transfer vector designed for insertion of foreign expression casettes in the polyhea ⁇ n locus of a baculovirus genome
• RT-PCR performed on total RNA isolated from EBV-transformed human spleen cehs, usmg a hB7 1 specific pnmerset (MR67/MR68) designed based on the hB7.1 se ⁇ uence, pubhshed by Freeman et al., 1989, as described in de Boer.M et al , 1992
• the amplified DNA was cloned m several eukaryotic expression vectors mcludmg pcDNAIneo (Invitrogen) for mammalian expression
• the resulting plasmid was named pcDNAIneohB7 1 (ICCG1713)
• the fuh size hB7 2 cDNA sequence was originally cloned by RT-PCR on human peripheral blood mononuclear cehs usmg hB7 2 specific pnmerset IG2834/IG2833
• the amplified fragment was mserted blunt end the EcoRV openened clonmg vector pBSK(+) for sequence confirmation, and later remserted in the mammalian expression vector pcDNA3 as an EcoRV-Xbal DNA fragment, resulting m pcDNA3hB7 2 (ICCG2307)
• the cDNA sequences codmg for the ED of hB7 1 and hB7 2 were then isolated by PCR on the above described plasmids pcDNAIneohB7 1 and pcDNA3hB7 2 containing respectively L ⁇ B7 1 and hB 7 2 fuh lenght cDNA, and usmg hB7 1ED or hB7 2ED-spec ⁇ fic p ⁇ mersets
• the resulting amplified DNA fragments were originally mserted in the clonmg vector pUCl 8 for se ⁇ uence confirmation of the inserts and later remserted m the pVL-Fc (ICCG3048) by BamHI-Bghl clonmg resulting in in frame fusion of the B7 1ED and B7 2 ED to the IgG ⁇ 1-Fc cDNA sequence already present in the pVL-Fc transfer vector resulting respectively the pVLshB7 1-Fc (ICCG300
• Soluble hB7. lglu-glu was expressed as a fusion protein consisting of the extracellular domain (AA1-242) of the hB7.1 protein carboxyterminahy fused to the peptide EEEEYMPME , also named glu-glu epitope, for which monoclonal antibodies are available.
• the hB7. lglu-glu encoding fusion cDNA sequence was generated by RT-PCR performed on total RNA isolated from an EBV-transformed human spleen ceh line, using a hB7.1ED specific primerset (MR67/ MR145) based on the hB7.1 sequence pubhshed by Freeman et al., 1989 , as described in de
• the sense primer matched with the codons for caninoacids 1 and 2 of the hB7.1 and contained a Pstl cloning site upstream this ED-coding sequence.
• the antisense primer matches amino acid-codons 203-242 of the hB7.1 sequence fohowed by the sequence encoding the glu-glu detection/purification epitope, and a Kpnl cloning site (figure 4).
• MR67sense primer Pstl 5' gcg ct ⁇ cag c ⁇ tctgaagcc atg ggc c 3'
• MR 145 antisense primer Kpnl 5' qc ⁇ c ⁇ atacc tta etc cat ggg cat gta ttc etc ttc ctg gtt ate agg aaa atg ctg ttg 3'
• the resulting 779bp PCR fragment was inserted in the baculo transfer vector pAcC8, a classical pAcYMl derived transfer vector (Matsuura et al.. 1987), as a Pstl- Kpnl fragment.
• the resulting pAcC8hB7.1 glu-glu transfer plasmid was used for insertion of the polyhedrin promoter controlled hB7. lglu-glu expression unit in the baculovirus polyhedrin locus by in vivo homologous recombination as described herunder.
• Soluble hB7.2his was expressed as a fusion protein of the extracellular domain (AA1-238) of B7.2 carboxyterminahy fused to a six histidine peptide.
• the hB7.2his tagged fusion cDNA se ⁇ uence was generated by PCR amplification performed on the plasmid pcDNAhB7.2(ICCG2307), containing the fuh size hB7.2 cDNA sequence as a template, using primerset IG8568 and IG8569.
• Antisense primer IG8569
• the recombinant transfer plasmids were introduced by classical transfection methods in msect cehs (Spodoptera frugiperda , Sf9) together with the linearized genomic DNA of a modified version of the wild type AcNPV baculovirus genome, the BaculogoldTM In this BaculogoldTM baculovirus genome the poiyhedrm gene is replaced by the E coh lacZ gene and contains three Bsu36I restriction sites side respectively the lacZ gene and the baculoviral genes ORF603 and ORF1629, originally flanking the polyhedrin locus.
• the hB7.1ED and hB7.2ED fusion proteins were secreted as a mature glycosylated monomer protein with M.W. ⁇ 62kDa (12.5% SDS-Laemmh, reducing conditions , Western blot analysis with anti-Fc mAb). In non-reducing conditions the proteins showed a high degree of ohgomerisation.
• Theoretical M.W. calculated for the non- glycosylated proteins is 53827 Da for hB7.2ED fusion protein and 54298 Da for hB7.1ED fusion protein. Optimal harvest time was determined at 72h post-infection for both proteins.
• the hB7.1ED glu-glu protein was secreted as a mixture of mature differentially glycosylated monomer proteins with M.W. _r 33kDa ( 12.5% SDS-Laemmh, reducing conditions , Western blot analysis with anti-glu-glu mAb).
• Theoretical M.W. calculated for the non-glycosvlated protein is 28768 Da.
• Optimal harvest time was determined at 72h post-infection. Total deglycosylation of tine protein was obtained with N-glycosydaseF.
• the hB7.2h ⁇ s protem v as secreted as a mixture of mature differentially glycosylated monomer proteins with M.W.
• Both B7.1ED and B7.2ED fusion proteins as weh as the BF.1ED glu-glu and the B7.2his proteins were analysed in a binding-competition test on an EBV transformed human B-ceh line RPMI8866 expressing hB7.1 and hB7.2.
• Dilutionseries of the crude infected ceh conditioned medium (CM) was incubated with a fixed, suboptimal concentration of respectively anti-B7.1 (B7-24)(20ng/ceh pehet) or anti-B7.2(lG10)(30 ng/ceh pehet) mAbs.
• the antigen/antibody mixture was subsequently incubated with the RPMI8866 cehs and anti-B7.1 or anti-B7.2 antibody binding on the cehs was then determined by Fluorochrome activated ceh scan (FACs) using fluorescein isothyocyanate (F ⁇ TC)-conjugated goat anti-mouse (Fab) antibodies .
• FACs Fluorochrome activated ceh scan
• F ⁇ TC fluorescein isothyocyanate
• Fab goat anti-mouse
• CM was stored at -70°C untih purification.
• B7 1ED and B7.2 ED fusion molecules were tested in binding inhibition studies, using anti-B7 1 mAb (B7-B7-24) and ant ⁇ -B7.2 mAb (1G10), on
• RPMI8866 cehs expressmg both B7 1 and B7.2 molecules.
• a suboptimal concentration of anti-B7 1 mAb (B7-B7-24) (20ng/ceh pehet) or of anti-B7.2 mAb ( 1G10) (30 ng/ceh pehet) was preincubated for 20' at 4°C with different concentrations of B7.1ED or B7.2ED fusion molecule (serial dilution ranging from 1 ⁇ g/ml to 2ng/ml) Cehs (0.5 x 10 6 cells/sample) were incubated for 20' at 4°C with the B7 1ED fusion molecule/B7-24 complexes or with the B7 1ED fusion molecule/lGlO complexes or with the B7.2ED fusion molecule/B7-24 complexes or with the B7.2ED fusion molecule/lGl O complexes JY cehs ( lxlO 5 cells/sample)
• the results show that a low concentration
• Purified B7.1ED glu-glu molecule was tested in ELISA , using anti-B7.1 mAb (B7- 24). Purified B7.1ED glu-glu molecule (0.5 ⁇ g/ml) was coated (400 overnight) on Nunc Maxisorb strips in carbonate buffer, pH 9.6. The experiment was further performed as described above. Results show that B7.1ED glu-glu molecule can bind anti-B7.1 monoclonal antibody (B7-24) at a coating concentration of 0.5 ⁇ g/ml.
• the anti-B7.1 mAb B7-24 and the anti-B7.2 mAb 1G10 are used for the generation of B7.1 and B7.2 scFv ' s respectively.
• Both the B7.1 and the B7.2 scFv's are used as positive controls in screening procedure for the generation of the B7.12 Mab.
• they serve as the B7.1-, B7.2-binding component in the bispecific diabody, the bispecific BiTAb and the trispecific triabody (see further).
• these scFv's are used as competitor reagents in the selection for high affinity variants of the anti-B7.12 scFv.
• Anti-B7.1 (B7-24) Mab VH and VL regions available in a baculo expression vector are previously described in WO 94/01547.
• both variable regions wee PCR-amphfied using the degenerate primers available from the Pharmacia RPAS (Recombinant Phage Antibody System) Mouse scFv Module.
• both VH and VL were linked using a short synthetic linker.
• de B7-24 scFv was ligated in pCANTAB5E.
• phages were generated after ligation in pCANTAB5E.
• TGI cells were transformed with the ligation mixture and were infected with M13K07 helper phage to induce phage production.
• the produced phages were panned upon a B7.1 positive B cell line (JY cehs), washed and subsequently eluted from the JY cehs. Binding phages were screened for their binding capacity in a FACS analysis with this B7.1 positive B ceh line. Co ⁇ ect binders were selected for further DNA sequence analysis. The sequence of B7-24 scFv clone 6 was found to be correct.
• VH and VL regions were cloned by RT-PCR with a set of degenerate forward primers located at the 5' end of the VH or VL region and a backward primer located in the constant region of the heavy or tight chain.
• the resulting cDNA was cloned in pUC18 and consensus VH and VL sequences were determined.
• VL and VH regions were cloned in the pCANTAB5E vector using degenerated primers obtained from Pharmacia, transformed in TG 1 cehs, infected with M 13K07 helper phage, fohowed by panning upon a B7.2 positive B ceh line (JY cells), as described for the anti-B7.1 regions.
• Correct binders were selected for further DNA sequence analysis. The sequence of I G I OSCFV clone 31 was found to be correct.
• the recombinant phages expressing B7-24 scFV( clone 6) and 1G10 scFv (clone 21) were tested in FACS experiments using FY cehs, expressing B7.1 and B7.2 molecules (de Boer et al., 1992). JY cells ( lx 10 5 cells/sample) were incubated for 20' at 4 C C with B7-24 scFv or 1G10 scFV phage diluted serial from undiluted to zero.
• the specific binding of the scFv phages is expressed as the mean fluorescence intensity in arbitrary units.
• the results showed that the B7-24 scFv and the lGlOscFv phages binds to the JY cehs.
• the mean fluorescence intensity (MFi) diminished when the phage is titrated.
• Evidence showing that the obtained scFv phages are specific for B7.1 or B7.2 was obtained in a competition FACS experiment on JY cehs, expressing B7.1 and B7.2 molecules.
• RPMI 1640 supplemented with 10% FCS the cehs were incubated for 20' at 4°C with B7-24 scFv or 1G10 scFv phage diluted serial from undiluted to zero. After washing twice in RPMI 1640 supplemented with 10% FCS, the cehs were subsequently incubated for another 20' at 4°C with a sheep anti-M13 monoclonal antibody, washed twice in RPMI1640 supplemented with 10% FCS and incubated for 20' at
• cDNA encoding the scFv's from the pCantab5E vector are transferred to an E. coli expression vector.
• the genes encoding the scFvB7-24H6 and scFvlGlOH ⁇ were isolated by PCR with primer 6999 and 7002 for scFvB7-24 and primer 7001 and 7002 for scFvlGlO, v/hereby specific restrictionsites were created.
• PCR fragments were cloned in the pGEM-T-vector (Promega) and were subjected to DNA sequence analysis. After cleavage with Xhol - Bsil 107 and Eco47III - Bstl 107(Biolabs) of P GEM-TscFvB7-24H6 and pGEM-TscFvlG10H6 respectively, the genes encoding scFvB7-24H6 and scFvlG10H6 were cloned in an E.coli expressionvector under control of the IPTG inducible lac-promotor .They are preceded by the pelB signal se ⁇ uence to obtain secretion into the periplasm.
• a C- terminal histidine-tag for purification purposes was included.
• the expression plasmids containing the scFv constructs were transformed in the E.coli expressionstrain JM83. (Knappik and Pl ⁇ ckthun 1995). An overnight culture was 20x diluted in LB+ lOO ⁇ g/ml amp+ 1% glucose and incubated at 28°C until an OD600 of 0.5 was reached. After removing the glucose of the medium, the culture was induced with 0. ImM IPTG and further incubated at 28°C during ⁇ 18h. To isolate soluble periplasmic proteins , the method described by Neu and Heppel (1965) was used.
• cehs were harvested by centrifugation and resuspended in ice cold shockbuffer (lOOmM Tris-HCl pH 7.4; 20% sucrose, ImM EDTA, pH8). After incubation on ice during 10' with occasional stirring, the mixture was centrifuged at l O.OOOrpm during 1,5'. The supernatans was removed and the pellet was immediately resuspended in ice cold distihed water. After incubation on ice during 10' with occasional stirring, the mixture was centrifuged at 14.000rpm and the obtained supernatans was the soluble periplasmic fraction.
• ice cold shockbuffer lOOmM Tris-HCl pH 7.4; 20% sucrose, ImM EDTA, pH8
• Binding specificity of the expressed scFv's is checked by ELISA experiments using the ED fusion proteins, and FACS analysis using EBV-transformed cehs
• Binding of scFv's to their target molecules is detected by means of a his-tag present on these scFv's.
• mouse anti-his mAb (1/1000 cone) is coated on the plate in PBS for 2h at 37°C.
• B7-24 scFv is added in a twofold serial dilution starting from l ⁇ g/ml to 15 ng/ml and incubated for lh at 37°C .
• the fuh size mAb B7- 24 was added as positive control (l ⁇ g/ml).
• the B7.1 ED fusion protein 500ng/ml
• B7-24 scFv was also tested and compared with the fuh size mAb B7-24 for its binding capacity to the human B7.1 molecule present on the RPMI8866 cehs, a EBV transformed B ceh line positive for hB7.1 and hB7.2.
• RPMI 8866 cehs were incubated with B7-24 scFv or, as control, with anti-B7.1 mAb (B7-24).
• Cehs (0.5 xlO ⁇ s ⁇ mple) were incubated for 30' at 4°C with B7-24scFv or with anti-B7.1 mAb (B7-24).After washing twice in RPMI supplemented with 10% FCS, the cehs were incubated for another 30' at 4°C with a mouse anti-His mAb. After washing twice in
• T cehs were purified out of whole heparinized blood on Ficoh-Paque (density 1.077, Pharmacia Biotech) density gradients.
• the peripheral blood mononuclear cehs (PBMC) present in the interface were washed three times in 40 ml of RPMIbic supplemented with 10% inactivated FCS. Subsequently, monocytes were removed by cold aggregation (Mentzer et al., 1986).
• PBMC peripheral blood mononuclear cells
• PBMC peripheral blood mononuclear cells
• Monocyte aggregates were allowed to sediment over a 15' period incubation on ice, and the non- aggregated cehs containing enriched T cehs and B cehs were carefully aspirated and centrifuged for 10' at 1200 rpm.
• T cehs were further enriched using Lympho- Kwik-T (One lambda Inc, Los Angeles, CA). This reagents contains a mixture of anti-monocyte and anti-B ceh mAbs and complement.
• Lymphocytes were resuspended in 3 ml Lympho-Kwik-T and the mixture was incubated for 45' at 37°C. Subsequently, cehs were resuspended in 0.5 ml of PBS and centrifuged for 2' at 2000 rpm and washed twice.
• Ceh pehet of 1 subconfluent falcon was dissolved in 800 ml RPMIbic and 200 ml Mitomycin C (250 mg/ml) and incubated for 40' at 37°C fohowed by two washes. Subsequently, cehs are suspended in 30 ml RPMIbic and incubated for 15' at 37°C fohowed by one additional wash step.
• the binding affinity of the scFv's is compared with that of Fab fragments of the parent antibody in order to evaluate whether the scFv fragments have the same affinity as the intrinsic affinity of the parent antibody, which is reflected in the binding characteristics of the Fab fragments.
• the ED fusion proteins are used as target molecules and the conditions for appropriate analysis of the different molecules are optimized for BIAcore analyses. These accumulated data give an indication of the extend with which the affinity is lowered by the construction of a scFv and set a goal for the increase in affinity obtained using Parsimonius mut agenesis (PM).
• This novel molecule is a single mAb that can bind to both B7.1 and B7.2.
• both B7.1-B7.2 ligands, CD28 and CTLA4 bind B7.1 and B7.2 with comparable affinities, it has not been possible to produce murine Mabs which react with both B7.1 and B7.2. This is probably due to the fact that the active B7.1- B7.2 epitopes, which are conserved between B7.1 and B7.2, are also sufficiently conserved between mouse and human to ensure that idiotypes against the human epitopes would be suppressed in the mouse as anti-self.
• mice are immunized with peptides derived from the homologous sites between B7.1 and B7.2.and, as an alternative route, human Mabs which react with high affinity' to both human B7 molecules are made via antibody engineering. Tv/o types of phage display libraries are used to obtain such antibodies by biopanning on immobilized B7.1 ⁇ D and B7.2ED fusion proteins.
• a ph ⁇ ge-displ ⁇ y conform ⁇ tion ⁇ l constrained library is screened which ends up in the development of small antigen binding peptides or fragments (also called microproteins) interacting with B7.1 and B7.2.
• CTLA4 elements are grafted onto a limited set of structurally homologous antibody scaffolds to define which elements within CTLA4 may be replaced without loosing B7.1-B7.2 binding. Otherwise, irnmunoglobulin sequences are grafted onto structurally and functionally important CTLA-4 sequences.
• CTLA4 is a member of the immunoglobulin superfamily, and as such its main extracellular region is buht up of a domain with a basic immunoglobulin fold.
• CTLA4-elements are grafted (CDR1-3 Including the 'MYPPPY" sequence, possibly FR residues), onto which scaffold.
• the 'scaffolds' are derived from the structurally most homologous antibody V-genes. For example, FR2 and FR3 are most homologous to human lambda tight chains with respect to amino acid sequence, with some homology for human kappa regions.
• Such chimaeric molecules are created and tested for binding to B7.1 and B7.2; this generates very useful information for the second stage of this approach.
• Large repertoires of selected antibodies (and their FR) are then made and are provided with a minimum of CTLA4-elements; these molecules are displayed on the surface of ph ⁇ ge. From these hybrid hbr ⁇ ries molecules that bind to both B7.1 and B7.2 are selected.
• the CTLA4 CDR regions are shuffled at the DNA level with the selected antibody FR sequences; oligonucleotides encoding the CTLA4 CDR regions fused to neighbouring antibody-derived framework residues are combined with 4 repertoires of frameworks (FR1-4) by PCR-assembly.
• Certain templates VH, V- kappa or V-lambda are chosen based on successful identification of closely homologous FR sequences.
• CTLA-4 on filamentous phage is a pre-requisite for the rational design approach using CTLA-4 as a scaffold.
• the PhagemidpCES 1 is represented : antibody genes : V L -C L , variable (V) and constant (C) region of the tight chain; V H -C H1 , variable and first constant region of the heavy chain; PlacZ, promoter; rbs, ribosome binding site; S, signal sequence; H6, six histidines stretch for IMAC purification; tag, c-myc-derived tag; amber, amber codon that allows production of soluble Fab fragments in non- suppressor strains, gill, gene encoding one of the minor coat proteins of filamentous phage. Restriction sites used for cloning are indicated.
• Clones with the correct sized insert were further tested by BsfNl fingerprint and shown to have the expected pattern.
• Phage were prepared and tested in ELISA for binding to B7-lIg and B7-2Ig.
• a strong signal was obtained showing binding to both B7.1-Ig and B7.2-Ig. and not to BSA or plastic .
• Soluble CTLA-4 also showed some binding to B7-1 and B7-2 in ELISA, however the signal was only twice background. It is interesting to see what the enrichment factor is of CTLA-4 phage for the ligands B7- 1 and B7-2, and to determine if it wih indeed be possible to select novel binding molecules.
• CTLA-4 binds to B7-1 and B7-2 with Kd of 0.4 and 2.2 ⁇ M respectively with a very fast off rate (koff > 0.4 sec _1 )(Greene et al., 1996 ; Van der Merve et al., 1997).
• the enrichment factor of CTLA-4 phage on B7-lIg and B7-2Ig was 5100 and 2687 respectively. This enrichment factor is very high , of the order 10 3 per round of selection and is presumeably due to the avidity effect of display on filamentous phage. This means that, provided chimeric CTLA-4/Vgene molecules are be expressed and displayed efficiently, that selection is possible even for lower affinity interactions than the wild-type CTLA-4/B7-12 interaction.
• a rational protein design can identify a structurally homologous protein scaffold (ie a member of the immunoglobulin superfamily (e.g V H , V L , CD2, CD 122, CD28) upon which we can graft structurally and function ally relevant regions of CTLA-4.
• a structurally homologous protein scaffold ie a member of the immunoglobulin superfamily (e.g V H , V L , CD2, CD 122, CD28) upon which we can graft structurally and function ally relevant regions of CTLA-4.
• the immunoglobulin hght chain A27(DPK22) is the most frequently used human hght chain in vivo and can be used as a scaffold.
• Light chains are generally more soluble than heavy chains, and light chains are more structurally compatible with CTLA-4 than heavy chains.
• VRVTV -CDR3 of CTLA-4 introduced at a structurally compatible site of A27 as KVEL-CDR3-
• the CK hght chain is appended to the C terminus as aKpnl/Notl fragment to solve the solubility problem, as expected with a unpaired tight chain molecule, .
• the generated molecules are manipulated in a rational way using modelling predictions to engineer changes to resolve any functional, structural or solubility difficulties. Furthermore this molecule is fed into the random approach.
• CiT-A-4/__mmunoglobulin molecules displayed on phage where we have amplified with primers specific for both the N and C-termini of immunoglobulin hght chains.
• CTLA-4 CDR3 mto hght chain repertoire we have designed primers which can be used to introduce the functionally important CTLA-4 CDR3 sequence into a immunoglobulin light chain repertoire at a structurally compatible position. These oligonucleotides are also used to spike DNA shuffling experiments
• Sequences of the CTLA-4 CDR3/Vk light chain spiking oligonucleotides are designed so as to amplify all VK sequences and are presnted in figure 12
• Sequences of CTLA-4 CDR3/ V ⁇ light chain spiking oligonucleotides are designed so as to amplify all V ⁇ sequences and are presented in figure 13
• This method selects antibodies binding to both B7.1 and B7.2 from a large ncrive Fab library, which contains 4.1 x 10 16 antibody molecules (Target Quest) displayed on phage. Selections are alternated between (biotinylated) B7.1ED and B7.2ED fusion protein and with excess of non-labeled Ig to deplete Ig-binding antibodies. This selection mode yields antibodies that target the (partially) overlapping binding site of CTLA4 on B7.1 and B7.2. It is also necessary to ensure the removal of antibodies specific for the junction between B7.1/2 and the Fc moiety; therefor alternating selections are done with KB7. lglu-glu and B7.2 his proteins, without the Fc portion.Gene diversification is required to improve the selectivity of the antibody. For this, the library quality is optimized using Trinucleotide-directed Mutagenesis (TRIM) technology from Morphosys (Virnek ⁇ s et al. 1994).
• TAM Trinucleotide-directed Mutagenesis
• This metinod selects sm ⁇ h antigen-binding peptides from random DNA sequence phage display libraries composed of cyclic peptides (Tn6, Tn8, Tn9, TnlO and HTS) and variants from human-origin proteins (Kunitz and endothelin libraries) ( described in US patent numbers 5,403,484 and 5,571,698 and 5,223,409 to Ladner et al.). These libraries allow to examine several hundred million compounds and useful 'hits' emerge. Both the structured peptide' hits' and the human origin 'hits' give rise to suitable clinical leads. To obtain ligands that bind both B7.1 and B7.2 the libraries are screened in three different ways:
• B7peptide 1 region 66-94 human B7.1
• B7peptide2 region 137-148 human B7.1
• B7peptide4 region 200-212 human B7.1
• Two peptides were derived from human B7.2: B7peptide3: region 124-135 human B7.2
• B7peptide5 region 183-194 human B7.2
• the peptides were synthesized on Tent ⁇ gel S resm (R ⁇ pp Polymere GmbH Germany) usmg a Rainin Symphony/Multiplex synthesizer with standard Fmoc-chemistry and HOBtiTBTU (TBTU 2-( lH-Benzotnazole-l -yl) -1, 1 3 3-tetramethyluromum tefrafluoroborate, HOBt N-Hydroxybenzotriazole) m situ activation Standard double couphngs were performed usmg a 4 fold excess of ammo acids the presence of equimolar amounts of HOBt and TBTU for 2 x 20 minutes
• the Fmoc protecting group was removed
• the peptide After completion of the peptide-resm complex the peptide is cleaved of the resm by incubating for 2 5 h with 90% t ⁇ fluoroaceuc acid/ 5% th ⁇ oamsole/3% ethaned ⁇ th ⁇ ol/2% amsole The peptide is precipitated from the cleaving mixture usmg t-burylmethylether After centnfugation the pellet is washed 3 times with t-butylmethylether and dried overnight under vacuum The purity of the crude peptide is checked on RP-HPLC
• B7 peptides were synthesized B7peptidel (IGP1458) Acetvl WQKEKKMVLTGGXGGEYKNRTIFD CONH2
• B7peptide2 (IGP1459) Acetyl LSVKADFPTPSI - CONH2
• B7peptide3(IGP1460) Acetyl LSVLANFSQPEI CONH2
• B7Pept ⁇ del B7pept ⁇ de2 B7pept ⁇ de3 and B7pept ⁇ de4 are biotiniiated peptides
• mice are immunized with these different peptides containing overlapping sites from B7 1 and B7 2
• a classical immunization scheme of 4 injections at 4 wee S interval is fohowed
• Bleedings are obtained at day 0 (pre immuun serum), day 66 (bleeding 1) and at day 94 (bleeding 2).
• Pre and post immuun sera are tested in ELISA for reactivity with B7.1 and B7.2 using the B7.1ED and B7.2ED fusion proteins.
• Mice developping antibodies reacting with both B7.1 and B7.2 are boosted and sacrificed 3 days after the boost.
• the fusion is performed using a standard protocol. Briefly, splenic cehs from the immune mouse are fused with SP2/0 myeloma cells in ration 10:3 using PEG/DMSO. Fused cehs are plated in 96-weh plates at density of 5 x 10 3 -10 4 cehs per weh and the first screening is performed 7 to 10 days later by ELISA for reactivity with B7.1 and B7.2. Positive clones are subcloned by limiting dilution and tested for reactivity with B7.1 and B7.2. Those hybridomas reacting with both B7.1 and B7.2 are selected for further characterization.
• the selected hybridomas are then tested for their affinity for B7.1 and B7.2 in BIAcore experiments. They are also tested in an in vitro system of humanT ceh-APC interaction, in order to show their efficiency as blocking agents for allogeneic T ceh activation, ability to induce anergy, and ability to exclude any direct stimulating effect on the APC.
• the efficacy of these mAbs as immunosuppressive agents is tested in in vitro models as outlined further.
• humanized versions of rodent Mabs has successfully been achieved by various methods.
• Humanized Mabs exhibit the same ligand- binding properties as the original rodent Mabs, but in general with severely decreased binding affinity.
• human Mabs (hulvlabs) derived from ncrive human libraries only have primary response-level affinities, i.e., sub-micromolar. However, such affinities are sthl 2-3 orders of magnitude below what is usually required for therapeutic efficacy, and affinity maturation will have to be completed m vitro.
• the affinity of the newly generated anti-B7.12 mAbs is maturated using m vitro mutagenesis techniques.
• the anti-B7.1 and anti-B7.2 scFv necessary for the different constructs (see further) are affinity maturated.
• In vivo antibodies are affinity matured in a stepwise fashion, by gradually incorporating mutations that cause small incremental improvement of the affinity
• the in vitro affinity maturation process essentially involves three steps, ( 1) the introduction of diversity in the antibody V-genes, creating a 'secondaryi library', (2) the selection of the higher affinity from the low affinity variants, and (3) the screening to allow discrimmation of antibody variants with differences in affinity or kinetics of binding.
• the selection is chosen to favour kinetic parameters such as off-rate or affinity; this hinges on the use of hmited and decreasing amounts of antigen, and on performing the selections in solution rather than by avidity-prone panning.
• antibodies of the highest affinity can be preferentially selected by using the antigen concentration at or below the desired dissociation constant.
• the areas that are best targeted for mutagenesis differ for each individual antibody. Diversity is introduced either more-or-less randomly, as with error-prone PCR, by using mutator strains, by V-gene chain shuffling, or by DNA shuffling. Chain shuffling is used successfully to obtain a 300-fold increase in the affinity of an anti-hapten antibody, originally isolated from a naive human scFv library.
• the antibody is CDR regions is targeted, using oligonucleotide directed mutagenesis or spiked oligonucleotides and PCR.
• CDR residues constituting the antigen combining site.
• residues that contact the antigen or may influence other residues contacting the antigen are targeted.
• residues are also determined experimentally, by chain shuffling, by alcmine-scanning of the CDR-regions, by parsimonious mutagenesis (see below), or by modelling.
• Residues involved in maintaining the main chain conformation of the CDR are conserved; residues that modulate affinity are randomized, ideally 4-6 residues at a time to allow efficient sampling of the sequence space. Sequential targeting is preferred, as additive effects of mutants obtained by targeting CDRis in parallel and combining mutations prove frequently unpredictable. For the different antibodies that are considered for affinity maturation, the following schedules are fohowed.
• the shuffle between CTLA-4 and antibody elements yields Fv-like molecules with an Ig-fold that bind to both B7.1 and B7.2.
• the affinity of these molecules is low, around 10 to 100 nM for the Kd.
• the maturation concerns antibody-like molecules, in which it remains unclear which areas of the molecule are involved in antigen binding. Therefore the first V-gene segment is exchanged via DNA-shuffling (basically similar to the first experiments, now with more defined subsets of V-gene derived segments), and in a parallel approach, the XLl-Red mutator strain.
• Both the heavy and hght chain CDR' ⁇ are targeted, in a sequential way, to derive antibody variants, which are than selected using limited amounts of biotinylated antigens (retaining parallel dual selections to retain affinity for both target. Initially both H3 and L3 are targeted; however, other CDR's also have to be diversified, because the binding site is extraordinary broad due to the dual binding capacity.
• the method of choice depends on the starting affinity, and fohow ⁇ the previous schedule (chain shuffle mutator strain) and as an alternative making use of spiked oligonucleotides to mutate H3 and L3.
• Affinity maturation in vitro is typically attempted by chain shuffling, or by random mutagenesis of the antibody CDRs and screening or selection for higher affinity variants. Either complete randomization or error-prone PCR is usually used as the mutational operator, but these are extremely inefficient probes for searching protein sequence space.
• Parsimonious Mutagenesis a proprietary computer-assisted method for otigonucleotide-directed scanning mutagenesis, called Parsimonious Mutagenesis (PM; Balint and Larrick, 1993; Schier et al., 1996).
• the efticiency of PM is based on a rational reduction in the size of the se ⁇ uence space which must be searched to one that can be completely encompassed in manageable libraries, and thoroughly searched with available screening techniques. This rational reduction of the sequence space is based on several observations. From the available X-ray cryst ⁇ hogr ⁇ phic data, between 15 and 22 residues of a protein antigen are typically in contact with a similar number of residues in the Ab combining site (Novotny et al., 1989; Laver et al., 1990; Novotny, 1991).
• the genetic code has evolved to maximize the frequency of adaptive substitutions among the 5-7 substitutions for each amino acid which may result from single base changes.
• the relevant sequence space for at least the first round of antibody affinity optimization as including all permutations of 1-5 mutations in up to 30 sites of the heavy and light chain CDR3s, and including ah possible combinations of one-base amino acid substitutions at each site. This comprises a sequence space of ⁇ 10 9 different sequences.
• PM it is possible to construct a library which would contain the entire sequence space in — 10 9 clones, and using phage display it is possible to pan the entire library against immobilized antigen.
• PMCAD a computer program
• a mutation frequency of 16.7% at each of 30 aa positions produces a binomial distribution in which 5-hit mutants comprise 19% of the library and mutants with 1-5 hits comprise 61% of the library.
• Substitute amino acid sets are controlled by using degenerate (doping) codons which may be selected to encode mixtures of more and less conservative analogs of the parent amino acid.
• NNT and NNG codons are particularly useful in this case because for most aa, they encode six different equally frequent 1-base codon changes to more and less conservative analogs.
• doping codons in PM the frequencies of 2- and 3-base codon changes is so low that the 1-base frequencies are only —5% below what they would be if they were the only substitutions.
• the overall com of the present invention is to generate a pharmaceutical composition that can simultaneously block the B7 1-CD28 and the B7 2-CD28 costimulatory pathways
• a BiTAb molecule can be constructed as an alternative to a diabody and a triabody molecule
• a BiTAb molecule has a less rigid structure compared to a diabody and a triabody, and cross-lmks and/or cross-reacts with the costimulatory molecules B7 1 and B7 2 expressed on the membrane of professional antigen- presenting cells, leading to the inhibition of antigen-specific T cell acuvation
• a BiTAb is a Bispecific Tetravalent Antibody molecule.
• the molecule consists of 4 scFv's; two anti B7.1 scFv's and two anti B7.2 scFv's (tetravalency).
• One single BiTAb is a homodimer of two identical molecules, each containing both an anti B7.1 and anti B7.2 scFv (bispecificity).
• An anti B7.1 and an anti B7.2 scFv are linked together using a dimerisation domain, which drives the homodimerisation of the molecule (see figure 14).
• VH and VL regions of the anti B7.1 and anti B7.2 Mab's are cloned from their respective hybridoma' s by RT-PCR with a set of degenerate primers, for example the ones used in the Pharmacia RPAS Mouse scFv module.
• both VH and VL are linked using a short synthetic linker.
• the scFv coding sequences are ligated in a scFv expressionvector such as pCANTAB5E (Pharmacia).
• phages wih be generated after ligation in pCANTAB5E.
• Binding phages are screened for their binding capacity in a FACS analysis with the respective B7.1 or B7.2 positive cell-lines. Correct binders are selected for further DNA sequence analysis.
• Anti B7.1 (B7-24) and B7.2 (IGIO) scFv's are used as building blocks to generate the B7.1/B7.2 BiTAbs using standard recombinant DNA techniques. A single BiTAb subunit starts with an anti-
• the dimerisation domain in its turn links C-terminahy to the anti-B7.2 scFv or the anti-B7. IscFv.
• a detection and purification tag is added at the extreme C-terminus of the molecule.
• the sequence coding for the dimerisation domain and the flanking linkers is made synthetically using the metinod described by Stemmer ei al. (1995). Herefor we considered the optimal codon usage for E.Coii-expression. This synthetic domain is subsequently linked to both the anti B7.1 and anti B7.2 (with tag) scFv's.
• dimerisation domain As tinkers betv/een the dimerisation domain and the scFv's we use either the widely used (G 4 S) 3 sequence (Hoogenboom er al, 1991) or the flexible and proteolysis-resistant truncated human IgG3 upper hinge region (Pack & Pl ⁇ ckthun, 1992).
• dimerisation domain we used a leucine zipper type of domain (Kostelny et al, 1992; de Kruif & Logtenberg, 1996) or the helix-turn- helix motif described by Pack et al. (1993).
• This human peptide shows a 'leucine zipper' dimerisation motif with limited homology to Fos Jun proteins (Duprez et al. 1997).
• a Cystein could be included to enhance stability.
• As the C-terminal detection and purification tag we use a hexahistidine sequence. For immunogenic reasons we decided to make BiTAb molecules both with or without a His ⁇ tag. The sequences are assembled in such a way that functional domains are easily replaceable using unique restriction sites present in the molecule.
• the BiTAb coding sequence is inserted in anE.co ⁇ expressionvector with an appropriate secretion signal such as pelB or ompA signal sequence and is placed under control of an inducible promotor such as P lac , ?,_._. or P L .
• the BiTAb expressionplasmid is introduced into suitable E.coli expressionstrctins such as JM83 or HB2151. Expression levels in the E.coli periplasm and extent of dimerisation is analysed using small-scale expression experiments. Co ⁇ ectly dimerised BiTAbs are purified from the E.coli periplasm using IMAC chromatography.
• the DNA sequence and the protein sequence of the described BiTAbB7-24- 1G10H6 are represented in figure 15 and 16, respectively and the sequences of
• BiTAb IGl 0-B7-24H6 in figure 17 and 18, respectively.
• Equal amounts ( l Opmoles) from each of the 10 ohgo's (primer 7624 to 7633) were combined and the mixture was subsequently diluted 100-fold in 50 ⁇ l PCR mix containing lOmM Tris-HCl pH9/ 2.2mM MgCl ⁇ 50mM KCl/ 0.2mM each dNTP / 0.1% TritonX- 100/ 1 U of T ⁇ q polymerase/ 0.1 U of Pfu polymerase.
• the PCR program consisted of 35 cycles at 50°C for 30s. The obtained gene was amplified in a second PCR reaction.
• the gene assembly reaction mixture was dhuted 40-fold in 100 ⁇ l PCR mix containing lOmM Tris-HCl pH9/ 2.2mM MgCl 2 / 50mM KCl/ 0.2mM each dNTP / 0.1% TritonX- 100/ 5U of Taq polymerase/ 0.1U of Pfu polymerase/ 2 outside primers (primers 7622 and 7623) at a concentration of 1 ⁇ M.
• the PCR program consisted of 23 cycles at 48°C for 30s.
• the gene-amplified DNA was purified and ligated in the pGEM-T-vector.
• the assembled DNA sequence was subjected to DNA sequence analysis.
• 7626 5' -ATACCTCCGGAGGTGAACTGGAAGAGCTGTTGAAACATCT- 3' 7627: 5' -GACCTT CAGCAGTTCTTTCAGATGTTTCAACAGCTCTTC - 3' 7628: 5' -GAAAGAACTGCTGAAAGGTCCGCGGAAAGGTGAACTGGAG - 3' 7629: 5' -TTCAGGTGC TCAGCAATTCCrCCAGTTCACCTTTCCGCG - 3' 7630: 5' -GAATTGCTGAAGCACCTGAAAGAGCTGTTGAAAGGTACCC -3'
• 7631 5' -ATGGGTAGTATCACCCAGGGGGGTACCTTTCAACAGCTCT - 3' 7632: 5' -CCCTAGGTGATACTACCCATACCAGCGGTCAGGTGCAACT -3' 7633: 5' -CGCGGAATTCGCGTTCGCGACTAGTTGCACCTGACCGCTGGT-
• ⁇ Spel restrictionsite was introduced just before the start of the gene encoding the VH-domain of the gene encoding scFvB7-24 and scFvlGlO. This is done by PCR with the appropriate primers (primers 8067 and 8075). The PCR fragment was ligated in the pGEM-T vector and subjected to DNA sequence analysis.
• BTTABB7-24-1G10H6 subunit For the construction of BTTABB7-24-1G10H6 subunit, three fragments were ligated in-phase, namely the gene encoding the N-terminal scFvB7-24, the gene encoding the HDH-domctin and the gene encoding the C-terminal scFvl Gl 0H6. In that way the gene encoding BiTAbB7-24-lG10H6 subunit, preceded by the pelB secretion signal, was cloned in an E.coli expressionvector under control of the lac-promotor.
• Fragment 1 pscFvB7-24H6 cleaved with XhoI-EcoRI
• Fragment 2 pGEM-THDH cleaved with Xhol-Spel Fragment 3: pGEM-TscFv 1 G 1 Os-e cleaved with Spel-EcoRI
• This expressionplasmid pBiTAbB7-24-lG10H6 was transformed in the expression strain JM83.
• n overnight culture of pBiTAbB7-24-lG10H6 in JM83 was 20x diluted in LB ⁇ lOO ⁇ g/ml amp+ 1% glucose and incubated at 28°C until an OD600 of 0.5-0.6 was reached. After removing the glucose of the medium, the culture was induced with 0. ImM IPTG and furtiner incubated at 28°C during ⁇ 18h. Periplasmic fractions were prepared as explained before. Only a very small amount was secreted into the periplasm and allmost ah the expressed protein formed insoluble cytoplasmic inclusion bodies. This was confirmed by NH 2 -terminal sequence analysis whereby the pelB secretion signal was still observed.
• BiTAbB7-24-lG10H6 could't be weh secreted into the periplasm.
• the gene encoding BiTAbB7-24-lG10H6 without a secretion signal was cloned under control of the pL-promotor and the obtained plasmid was transformed to the E.coh expressionstrain MC1061(pAcI), SG4044(pAcI), UT5600(pAcI). After induction, cytoplasmic inclusion bodies will be formed and purification from inclusion bodies wih be performed.
• This human peptide shows a 'leucine zipper' dimerisation motif with hmited homology to Fos/Jun proteins.
• the gene assembly reaction mixture was diluted 40-fold in 100 ⁇ l PCR mix containing 1 OmM Tris-HCl pH9/ 2.2mM MgCl 2 / 50mM KCl/ 0.2mM each dNTP / 0.1% TritonX-100/ 5U of Taq polymerase/ 0.1U of Pfu polymerase/ 2 outside primers (primers 8526 and 8527) at a concentration of 1 ⁇ M.
• the PCR program consisted of 30 cycles at 50°C for 30s.
• the gene-amplified DNA was purified and ligated in the pGEM-T-vector.
• the assembled DNA sequence was subjected to DNA sequence analysis.
• the dimerisation domain HDH in the BiTAb molecule wih be replaced by this human dimerisation domain to reduce immugenecity.
• Diabodies are dimeric antibody fragments.
• a heavy- chain variable domain V H
• V L light-chain variable domain
• each antigen-binding site is formed by pairing of one V H and one V L domain from the two different polypeptides. This is achieved by shortening the linker between the V H and V L domains in each molecule (Holtiger et al., 1993). Since diabodies have two antigen-binding sites they can be bispecific.
• Mono- or bi-specific bivalent molecules are generated by shortening the flexible linker se ⁇ uence between the VH and VL of the anti-B7.1 scFv B7-24, the anti-B7.2 scFv IGIO and the scFv molecule with dual specificity for B7.1 and B7.2 (B7.12) to between five and ten residues (Gly4Ser to (Gly4Ser)2) and for the bi-specific molecules by cross-pairing the variable heavy and tight chain domains from the tv/o scFvs with different antigen recognition (B7.1/B7.2 and B7.12/B7.12).
• v/e have documented the construction of a anti-B7.1 diabody B7-24.
• the linker in scFvB7-24 was reduced to 5 or 10 aminoacids.
• the VH domain of scFvB7-24 was amplified with primers 8218 and 8070( 5aa) ,and the VL domain of scFvB7-24 with primers 8073 and 8075 .
• a Sapl restriction site was introduced at the N-terminus of the VH-domctin and a EcoRI restriction site was introduced at the C-terminus of the VL domain.
• Both PCR fragments were annealed and amplified by making a mix of equal amounts of both fragments, primers 8218 and 8075, 200 ⁇ M dNTP, 1U Pfu- polymerase and 1 Ox Pfu-buffer.
• the annealed fragment of 804 bp was ligated in the pBSK(+) and subjected to DNA sequence analysis.
• Ah 4 expressionplasmids were transformed to JM83.
• An overnight culture was 20x diluted in LB+ lOO ⁇ g/ml amp+ 1% glucose and incubated at 28°C until an OD600 of 0.5 was reached. After removing the glucose of the medium, the culture was induced with 0. ImM IPTG and further incubated at 28°C during ⁇ 18h.
• Periplasmic fractions were prepared as explained before. Only a small amount (10-30%) was secreted into the periplasm
• 8073 S'GTCACCGTCTCCTCAGGCGGAGGTGGCTCTGACATCGAGCTCACTCAGTCTC C -3' 8074:
• the produced bivalent monospecific scFv B7.24 diabody molecules are purified from the periplasmic extract .
• the bispecific diabodies (figure 27,28,29,30) are constructed otin a simhlar way as described for the monospecific diabodies.
• the diabody construct can target only two molecules being B7.1 and B7.2 or
• a construct which could target three molecules, e.g two B7.2 and one B7.1 molecule involved in this costimulatory pathway, namely a triabody, is aimed in this invention.
• a triabody is a mono-, a bi- or a trispecitic molecule recognizing simultaneously e.g two B7.2 and one B7.1 molecules. Similar to the diabody, the triabody is a molecule with a rigid structure that prevents simultaneously binding to the three targets and so prevents the activation of the APC.
• Each antigen-binding site is formed by pairing of one V H and one V L domain from the same or from two different polypeptides.
• Both PCR fragments were annealed and amplified by making a mix of equal amounts of both fragments, primers and , 200 ⁇ M dNTP, 1U Pfu-polymerase and lOx Pfu-buffer.
• the annealed fragment of bp was ligated in the pBSK(- ⁇ -)-vector and subjected to DNA sequence analysis.
• Both expressionplasmids, pTrcB7-24L0H6 and pTrclG10L0H6 were transformed to JM83.
• Periplasmic fractions were prepared as explained before. Only 5 a small amount ( 10-30%) was secreted into the periplasm
• bispecific triabodies are constructed in a similar way as described for the monospecvific triabodies (figure 35,36 and37,38).
• the multimeric behaviour of the purified and unpurified molecules is analyzed.
• the ability of the molecules to bind the different antigens is tested in ELISA, FACS, T 0 ceh proliferation assay and BIACORE as described under section 7.5.
• ScFvM24 L0 (PI and P2).
• PI from both constructs contained the pe ⁇ kfr ⁇ ctions with the highest protein concentration but also with the highest amount impurities.
• P2 (ScFvM24 LO) and P3 (ScFv M24 L5) contained the tailing fractions of the elution peak, low in protein concentration but with a higher ScFv purity.
• P2 from ScFvM24 L5 contained the fractions in between PI and P3 with an 5 intermediate purity. Elution fractions were not as pure compared to the earlier native purification performed with ScFvM24.
• Binding specificities of the unpurified monospecific scFvB7-24, B7-24 diabody (scFvB7-24 L5) and B7-24 triabody (scFvB7-24 LO) molecules and of the unpurified monospecific scFvlGl O, 1G10 diabody (scFvlGlO L5) and triabody (scFvlGlO LO) molecules were analysed in ELISA experiments using the B7.1ED and B7.2ED fusion proteins.
• scFvlGlO LO As the periplasmic preparations of scFvlGl O and IGI O triabody (scFvlGlO LO) contained very low amounts of scFv as demonstrated on Western blot, these two extracts were firstly concentrated 3.5 times before analysis in ELISA.
• rabbit anti-human IgG Fc fragment specific antibodies were coated on MaxiSorb plates at a concentration of 1 mg/ml in PBS buffer for 2h at 37°C or overnight at 4 3 C followed by blocking vith PBS 0.1% casein for lh at 37 D C.
• the unpurified monospecific scFvB7-24, B7-24 diabody and B7-24 triabody molecules were further analysed for their binding capacity to the B7-1 molecules on the membrane of mouse 3T6-CD32-B7.1 cehs, which are transfected with the cDNA encoding human B7.1.
• the 3T6-CD32 cehs and 3T6-CD32 cehs transfected with the cDNA, of human B7.2 (3T6-CD32-B7.2) were included as a negative control.
• binding of these molecules on B7.1 expressing EBV transformed human B cehs, RPMI 8866 was evaluated.
• FACS buffer PBS supplemented with 5% inactivated FCS and 0.02% sodium azide
• Periplasmic preparation of nontransformed JM83 cehs at the same dilutions as described above was included as a negative control.
• cehs were incubated with mouse anti-his monoclonal antibody at 2 mg/stctintiig for 20' at 4°C fohowed by 3 washes with FACS buffer.
• cehs were incubated with 1.4 mg/staining of fluorescein (FTTC)-conjugated goat anti-mouse IgG Fc fragment specific antibodies for 20' at 4°C, followed by three wash steps and analysis of the cehs for fluorescent stctinting using a FACScan instrument.
• FTTC fluorescein
• Binding of these recombinant antibodies was found to be specific for B7.1 as they did not bind to 3T6-CD32 and to 3T6-CD32-B7.2 cehs, which do not express B7.1 (data not shown). Binding specificities of the semi-purified monospecific B7-24 diabody
• scFvB7-24 and semi-purified B7- 24 diabody (scFvB7-24 L5 fraction p3) or B7-24 triabody (scFvB7-24 L0 fraction p2) molecules were added in a twofold serial dilution starting from 500 ng/ml to 7.5 ng/ml and incubated for lh at 37°C. After three washes with PBS 0.05% Tween20, wells were incubated for lh at 37°C with mouse anti-his monoclonal antibody fohowed by three washes and incubation for lh at 37°C with biotinylated goat anti- mouse (IgG +IgM) antibodies at 1.5 mg/ml.
• IgG +IgM biotinylated goat anti- mouse
• Binding specificities of the semi-purified monospecific B7-24 diabody (fraction p2 and p3) and triabody (fraction p2) molecules were also evaluated in a second ELISA.
• wehs were coated with mouse anti-his monoclonal antibodies ( 1 mg/ml) in PBS for 2h at 37°C or overnight at 4°C fohowed by blocking with PBS 0.1% casein.
• Results of the first ELISA setting show that the semi-purified fractions of B7-24 diabody (scFvB7-24 L5 p3) and of scFvB7-24 triabody (scFvB7-24 L0 p2) displayed binding to indirectly coated B7.1ED fusion protein, comparable with the binding of purified scFvB7-24. Similar results were obtained in the second ELISA setting.
• B7-24 diabody and B7-24 triabody display binding to B7.1ED fusion protein. The observed binding was specific as no significant binding was observed to the B7.2ED fusion protein.
• the cehs were incubated with fourfold serial dilutions of purified scFvB7-24 semi-purified B7-24 di ⁇ body (scFvB7-24 L5 fraction p2 and p3) or semi-purified B7 24 triabody (scFvB7-24 LO fraction p2), starting from 1 mg/ml for 20' at 4°C
• scFvB7-24 LO fraction p2 semi-purified B7 24 triabody
• T cells were purified out of whole hepar nized blood on Ficoh Paque (density 1 077 Pharmacia Biotech) density gradients
• the peripheral blood mononuclear cehs (PBMC) present m the t erf ace were washed three times m 40 ml of RPMIbic supplemented with 10% inactivated FCS Subsequently monocytes were removed by cold aggregation (Mentzer et al 1986) To this end PBMC '- r ere resuspended in 40 ml oi RPLIIbic supplemented with 10% inactivated FCS ana slowly rotated lor 30 at 4 C Monocyte aggregates were allowed to sediment over a 15 period incubation on ice ana the non aggregated cells containing enncned T cehs and B cehs were carefully aspirated and centrifuged for lO at 1
• Lympho Kwik T and tne mixture was incubated for 45' at 37 ⁇ C Subsequently cehs were resuspended in 0 5 ml of PBS and centrifuged for 2' at 2000 rpm and washed twice
• the mouse fibroblast cell tine, 3T6, tranfected with the cDNA encoding human B7_ 1 and human CD32 (3T6-CD32-B7 1) was mitomycin C treated Cell pellet of 1 subconfluent falcon was dissolved in 800 ml RPMIbic and 200 ml Mitomycm C (250 mg/ml) and incubated for 40' at 37°C followed by two washes Subsequently, cells are suspended m 30 ml RPMIbic and incubated for 15' at 37 D C fohowed by one additional wash step In the mixed lymphocyte culture (MLR), the mitomycm C treated 3T6-CD32-B7 1 cehs (10 4 cehs/weh) were incubated with OKT3 (1 mg/ml) for 1 h at 37°C fohowed by a 1 h incubation at 37°C with threefold serial dilutions of B7-
• MLR mixed lymphocyte culture
• mice anti-his antibody 1 mg/ml was coated in PBS for 2h at 37 " C fohowed by blocking with PBS 0 1% casern for lh at 37 " C
• the different gelfiltration fractions of scFvB7-24 B7 24 diabodies (scFvB7-24 L5 p3) and B7 24 tnabodies (scFvB7-24 L0 p2) were added twofold serial dilutions and indicated for lh at 37 C fohowed by incubation with 100 ml of B7 1ED fusion protem (500 ng/ml) for lh at 37°C Subsequently the wehs were mcubated with peroxidase-conjugated sheep anti- human Ig (whole antibody) at dt
• T lymphocytes and induces IFN- ⁇ release by Th-1 lymphocytes this stronger inhibition by the crosstinked anti-B7 1/B7 2 mAbs, compared to the inhibition by the combination of the two mAbs alone, means indirectly a stronger inhibition of the T lymphocyte activation
• these experiments demonstrate a stronger immunosuppression potency of the chemical crosstinked anti-B7 1/B7 2 mAbs compared to the combination of an anti-B7 1 mAb and an ant ⁇ -B7 2 mAb
• the crosstinked monoclonal antibodies were further evaluated to block T cell-APC mteractions in a MLR wherein T cells are cultured vith RPMI 8877 cells a EBV tranformed B ceh hne expressmg human B7 1 and human B7 2 molecules T cehs were purified out of whole hepa ⁇ nized blood on
• PBMC peripheral blood mononuclear cehs
• Lympho-Kwik-T Lympho-Kwik-T and the mixture was incubated for 45' at 37°C. Subsequently, cells were resuspended in 0.5 ml of PBS and centrifuged for 2 at 2000 rpm and washed twice.
• the mitomycin C treated RPMI8877 cells 10 4 cehs/weh were incubated with OKT3 ( 1 mg/ml) for 1 h at 37°C followed by a 1 h incubation at 37°C with serial dilutions of crosshnked
• B7.24/1G10 mAbs fraction 19 or 20 or with the combination of B7.24 ( 1 ⁇ g/ml) and 1G10 (l ⁇ g/ml).
• purified T cehs (5x10 4 cell/well) were added and incubated for 5 days.
• cehs were incubated with 1 mCi ( 3 H0thymidine for 6 h and harvested using an automated ceh harvester.
• 3 H-thymidine incorporation was determined with a liquid scintillation counter. Results showed
• a combination of anti-B7.1 mAb, anti-B7.2 mAb and CsA seems to be an optimal treatment to induce donor-specific tolerance during allo-transplant ⁇ tion.
• Rhesus monkeys receive a kidney transplantation from an allogeneic donor mismatched for at least one MHC class I and one class II antigen.
• Graft survival time is indicative for the immunosuppressive potency of the therapy combining anti-B7.1 mAb, ⁇ nti-B7 2 mAb and CsA
• the mAbs (0 5 mg/kg at day - 1 0 25 mg/kg on day 0 till 12) are given daily for 14 days starting at day -1
• the CsA ( 10 mg/kg) is given daily for 35 days starting at day 1
• At several time pomts durmg the experiment blood samples are taken to determine the blood level of CsA serum levels of the mAbs anti-B7 1 and anti-B7 2, the rhesus monkey anti-mouse anti-body (RAMA) response anti-donor antibody reponse
• B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366:76.
• CD2 is tinvolved in maintenance and reversal of human ahoantigen- specific clonal anergy. J. Exp. Med. 180:1665.
• B-ceh suriace antigen B7 provides a costimulatory signal that mduces T cells to proliferate and secrete mterleukm 2 Proc Natl Acad Sci USA 88 6575
• Costimulator B7- 1 confers antigen-presenting-cell tunction to parencnymal tissue ana m conjunction with tumor necrosis factor alpha leads to autoimmunity in trans gemc mice Proc Natl Acaa Sci USA 91( 1 1) 5138
• CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location. /. Immunol. 147: 1037.
• T ceh proliferation involving the CD28 pathway is associated with cyclosporine- resistant interleukin 2 gene expression. Mol. Cell. Biol. 7:4472.
• T-ceh antigen CD28 mediates adhesion with B cehs by interacting with activation antigen B7 BB-1. Proc. Natl. Acad. Sci. USA 87:5031.
• ICM-1 intercellular adhesion molecule- 1
• LFA-1 lymphocyte function-associated antigen
• Miniantibodies use of amphipathic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in
• T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3. Nature 326:400.
• CD28 activation pathway regulates the production of multiple T-ceh derived lymphokines/cytokines. Proceedings of the National Academy of Sciences of the United States of America 86: 1333.
• T- ceh activation by the CD28 ligand B7 is required for cardiac allograft rejection in "vivo. Proceedings of the National Academy of Sciences of the United States of America 89: 1 1 102.
• Atioantigen-specific anergy is induced by anti-B7 and cyclosporin A /. Exp. Med. 179:715
• Trinucleotide phosphoramidites ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Research 22(25):5600.

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