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WO1994005779A1 - Procede de verification de reponse immune individuelle a un anticorps a l'aide d'une souris a scid - Google Patents

Procede de verification de reponse immune individuelle a un anticorps a l'aide d'une souris a scid Download PDF

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Publication number
WO1994005779A1
WO1994005779A1 PCT/GB1993/001903 GB9301903W WO9405779A1 WO 1994005779 A1 WO1994005779 A1 WO 1994005779A1 GB 9301903 W GB9301903 W GB 9301903W WO 9405779 A1 WO9405779 A1 WO 9405779A1
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WIPO (PCT)
Prior art keywords
cells
mab
pbl
antibody
mice
Prior art date
Application number
PCT/GB1993/001903
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English (en)
Inventor
Alec Sehon
Soji Bitoh
Original Assignee
O'brien, Caroline, Jane
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB929219063A external-priority patent/GB9219063D0/en
Priority claimed from GB929226009A external-priority patent/GB9226009D0/en
Application filed by O'brien, Caroline, Jane filed Critical O'brien, Caroline, Jane
Priority to AU49770/93A priority Critical patent/AU4977093A/en
Priority to EP94908820A priority patent/EP0662127A1/fr
Publication of WO1994005779A1 publication Critical patent/WO1994005779A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells

Definitions

  • the present invention relates to materials and
  • xenogeneic molecules such as proteins.
  • Therapies based on xenogeneic proteins have rapidly expanded in recent years as a result of the increasing number of recombinant proteins, synthesized by new methods of molecular biology or produced by hybridoma technology.
  • xenogeneic proteins have rapidly expanded in recent years as a result of the increasing number of recombinant proteins, synthesized by new methods of molecular biology or produced by hybridoma technology.
  • xenogeneic proteins one may cite diverse enzymes, various biological response modifiers (BRMS ) such as lymphokines and hormones, murine monoclonal antibodies or recombinant antibodies directed to specific determinants of human T cells such as CD3, CD4 or IL-2 receptors, or of adhesion molecules.
  • BRMS biological response modifiers
  • mAbs monoclonal antibodies
  • mAbs monoclonal antibodies
  • mAbs monoclonal antibodies
  • mAbs monoclonal antibodies
  • the xenogeneic proteins referred to above have been shown to be potentially useful additions to the therapeutic armamentarium in a number of diseases, their effectiveness is undermined by their inherent immunogenicity. This is so even where the mAbs have been humanised.
  • the patient's production of antibodies against the xenogeneic treatment protein eg a murine mAb prevents or reduces its desired effector function.
  • the resulting immune complexes often lead to untoward pathophysiological effects, including serum sickness and occasionally even anaphylaxis.
  • UK Patent No. 1,578,348 and US Patent No. 4,261,973 disclose that an allergen (AL) such as ovalbumin and the non-dialyzable constituents of the aqueous extract of ragweed pollen and dog albumin, may be converted to a tolerogen by coupling it to an optimal number (n) of monomethoxy polyethylene glycol (mPEG) molecules.
  • A allergen
  • mPEG monomethoxy polyethylene glycol
  • the patent discloses that injection of a tolerogenic conjugate of human IgG into mice, prior to administration of conjugates of human IgG with either dinitrophenyl DNP or DNP-keyhole limpet haemocyanin (KLH) led to the abrogation of the capacity of the mice to mount humoral antibody responses to both human IgG and the conjugated moiety DNP or DNP-KLH. If however DNP-KLH was injected into mice pretolerized with a noncovalent mixture of DNP-KLH and a PEG conjugate of human IgG, the mice mounted normal humoral, antibody responses to DNP and KLH, but remained suppressed to human IgG.
  • DNP-KLH DNP-keyhole limpet haemocyanin
  • non-immunogenic mPEG conjugates of bovine adenosine deaminase are being repeatedly injected into children with severe combined immuno-deficiencies
  • HAMA human anti-mouse Ig antibody
  • hu-PBL-SCID mice represent the closest recently developed in vivo model of the human lymphoid system.
  • the present application concerns discoveries and ideas going on from the prior art reported above and technical applications based thereupon.
  • the application discloses that treatment of hu-PBL-SCID mice with a tolerogenic covalent conjugate of mPEG and an anti-ovalbumin IgG1 murine mAb (Mab-2), suppressed the human anti-mouse Ab responses to both the common ( ⁇ 1, ⁇ ) and the idiotypic determinants of Mab-2.
  • the Mab-2(mPEG) 36 conjugate suppressed the immune responses of hu-PBL-SCID mice to the common and idiotypic determinants of murine mAbs to the 2,4-dinitrophenyl residue and to human CD4, consisting also of ⁇ 1 and ⁇ chains.
  • a tolerogenic mPEG conjugate of a murine mAb induces pan-suppression of the human lymphoid system with respect to other murine mAbs that share the isotypic determinants of the original mAb incorporated in the conjugate.
  • anti-mouse antibody responses to any murine IgG mAb would be suppressed by one of eight mPEG conjugates, each incorporating one of the four subclasses of IgG and one of the two light chains.
  • the substance eg mAb, AL, BRM in the mPEG need not be exactly the same as the substance for which immunosuppression is sought, but only needs to have structural elements in common.
  • the present application provides a tolerogen for administering to a patient in order to reduce the patient's immune response to a given antigen (eg antigen 'X'), which tolerogen comprises a water- soluble covalent conjugate of another antigen (eg antigen Y) with one or more non-immunogenic water-soluble antigen (eg antigen Y) with one or more non-immunogenic water-soluble antigen (eg antigen 'X')
  • tolerogen comprises a water- soluble covalent conjugate of another antigen (eg antigen Y) with one or more non-immunogenic water-soluble
  • the given antigen and the another antigen may both comprise at least part of a xenogeneic protein.
  • the xenogeneic proteins may be antibodies.
  • the antibodies may be rodent.
  • the antibodies may be murine.
  • the antibodies comprising the given antigen and the another antigen may share the same isotypic determinants.
  • the antibodies comprising the given antigen and the another antigen may have different idiotypes.
  • kit which comprises a panel of eight different tolerogens as described above. There will be first to fourth tolerogens comprising antibodies with kappa light chains and of isotypes IgG1, IgG2, IgG3 and IgG4 respectively and fifth to eight tolerogens comprising antibodies with lambda light chains and of isotypes IgG1, IgG2, IgG3 and IgG4 respectively.
  • the water soluble polymer may be selected from the group consisting of poly(alkylene-glycols),
  • the polymer may be poly(alkylene glycol) or its monomethoxy derivative.
  • the polymer may be poly(ethylene glycol) or its monomethoxy derivative.
  • the water soluble polymer is poly(ethylene glycol) it may have a molecular weight in the range of 2000- 35,000. Preferably the molecular weight may be in the range of 3000-6000.
  • the patents may be human.
  • tolerogens have been disclosed for the purpose of suppressing a patient's immune response against a xenogeneic protein eg a mAb in order that the mAb may attack an undesired pathogen or tissue eg a tumour.
  • a xenogeneic protein eg a mAb
  • the applicants have been disclosed for the purpose of suppressing a patient's immune response against a xenogeneic protein eg a mAb in order that the mAb may attack an undesired pathogen or tissue eg a tumour.
  • Treatment Abs may for example, be used which are specifically directed against a mediator of the patent's immune response against an allograft.
  • the body may also mount an immune response against the treatment Ab which are themselves xenogeneic. Therefore a tolerogenic conjugate of the treatment Ab can be used to suppress the immune response against the treatment Ab, which is therefore able to act against the mediator and reduce the patient's immune response against the eg allograft.
  • the present application provides a tolerogen for administering to a patient in order to reduce their immune response to a treatment antibody or antibody binding fragment which is specific for an epitope of a mediator of the patient's immune response against a xenogeneic epitope, which tolerogen comprises a water- soluble covalent conjugate of one or more non-immunogenic water-soluble polymers with said treatment antibody or antibody binding fragment.
  • the xenogeneic epitope may be presented by an allograft.
  • the allograft may be a heart.
  • the mediator may be a cytotoxic T lymphocyte.
  • the epitope may be part or all of the T cell receptor.
  • the treatment antibody or antibody binding fragment may be rodent derived. It may be murine derived.
  • the treatment antibody may be anti-clonotypic. Also provided are pharmaceuticals comprising
  • tolerogens as described with one ore more excipients.
  • treatment substance eg a protein.
  • Tolerogens as
  • ⁇ may be used in the treatment of patients, particularly human patients, where it is desired to control immune responses against any xenogeneic material.
  • patients particularly human patients
  • immune responses against any xenogeneic material For example to treat patients to prevent allograft rejection or to treat patients to prevent or limit the immune response mounted against a treatment substance eg a protein.
  • hu-PBL-SCID mice represent an in vivo model of the human immune system.
  • a SCID mouse may be engrafted with PBL of a particular patient thereby providing that patient with their own personalised in vivo model of their immune system.
  • any tests which involve a monitoring of that particular patients immune response may first be tried out on their personal in vivo model.
  • This is of enormous benefit, not least because it minimises patient trauma.
  • the present application also provides a method for testing a patient's (P) individual immune response to antigen 'X' which comprises the steps of:
  • the method may also comprise treating said test mouse with a tolerogen designed for administration to the patient to reduce the patient's immune response to antigen 'X'.
  • the tolerogen may comprise a water-soluble covalent conjugate of antigen 'X' with one or more non-immunogenic water-soluble polymers.
  • the PBL sample may comprise both T cells and B cells.
  • the PBL sample may be enriched for B cells.
  • the PBL sample may contain approximately 20xl0 6 T cells and 20 ⁇ 10 6 B and MN cells.
  • the patient antibody response in the test mouse may be compared to a patient antibody response mounted in a control mouse which comprises a further p-PBL-SCID mouse treated with suitable control material, for example PBS.
  • the method may comprise detecting a patient antibody response which is specific to said antigen 'X'.
  • the sample of PBL may be substantially free of erythrocytes.
  • the patient antibody response may be detected by immunoassay.
  • the immunoassay may be an enzyme
  • the antigen 'X' may comprise at least part of a xenogeneic protein.
  • the xenogeneic protein may comprise an antibody.
  • the antibody may be of rodent.
  • antibody may be murine.
  • the antibody may be specific for a target antigen in the patient.
  • the target antigen may comprise a part or product of a pathogen.
  • the target antigen may comprise a part or product of the patient's cell.
  • the patient may be human.
  • SCID mouse for use in a method as described above which has been engrafted with a sample of PBL from a particular patient.
  • the method for testing a patient's individual immune response using a tailorised mouse model of their immune system can be used to test out an immune response against any treatment material eg mAb and to establish the effect of a tolerogen in reducing that immune
  • the treatment mAb could be for control of the patient's own immune response against a desired but xenogeneic material eg an allograft.
  • the mAb in the tolerogen may be different to the treatment mAb provided they have structural elements eg isotypic determinants, in common.
  • the present application also provides a method for testing a patient's (P) individual immune response to an antibody 'X' or a binding fragment thereof, which is specific for an epitope of a mediator of the patient's immune response against an antigen 'Z', which method comprises the steps of: (i) isolating a sample of
  • peripheral blood leucocytes from the patient; (ii) engrafting a mouse having severe combined
  • SCID immunodeficiency
  • tolerogen comprises a water-soluble covalent conjugate of an antibody 'Y' with one or more non-immunogenic water soluble polymers and wherein said antibody 'Y' although different from antibody 'X' has structure in common with it.
  • Figure 1 shows the relationship between human IgG level and HAMA response in hu-PBL-SCID mice.
  • Twenty-three 3 to 4-week old SCID mice were engrafted i.p. with 20 ⁇ 10 6 T and 20 ⁇ 10 6 (B+MN) cells on day 0. Eleven of the mice were injected with the tolerogen, Mab-2(mPEG) 36 , and the remaining 12 mice were injected with PBS prior to immunization with 20 ⁇ g of ha-Mab-2 on days 8 and 28.
  • Each hu-PBL-SCID mouse was bled on day 42 and the sera were assayed for total human serum IgG levels and HAMA response; each point on the graph represents the relationship between these two parameters with respect to one of the test and control mice (represented, respectively, by circles and squares).
  • Figure 2 shows a flowchart for the protocol for testing transferable suppression in hu-PBL-SCID mice
  • FIG. 3 shows a flowchart for the protocol for generation of responder CD4 + T cells.
  • Each SCID mouse received 50 ⁇ 10 6 PBL of each volunteer on day 0. All mice were immunized 12 hr later with 100 ⁇ g of Mab-2 emulsified in FCA. Two weeks later, human leucocytes were isolated from pooled spleens and lymph nodes of each group of 2-4 hu-PBL-SCID mice and treated with anti-H-2 d mAbs and RC. The cells were stimulated twice with 100 ⁇ g/ml of Mab-2 or 20 ⁇ g/ml of PPD at 21 days interval. Two weeks after the last stimulation, the culture cells were treated with OKT8 plus RC'. The residual viable cells were used as responder CD4 + T cells.
  • Figure 4 shows suppression of HAMA to the idiotype of murine anti-OVA monoclonal Mab-2 by Mab-2(mPEG) 36 conjugate.
  • Panels A and B hu-PBL-SCID mice injected with either 200 ⁇ g of Mab-2(mPEG) 36 (circles) or PBS (squares) were immunized with 1 ⁇ g of Mab-2 in FCA 7 and 21 days later, and were bled 21 days after the second
  • Panel A Each serum was titrated in the presence of a mixture of biotinylated and non-biotinylated Mab-2; Panel B: Each serum titrated in the presence of a mixture of biotinylated and non-biotinylated Hl-DNP ⁇ -109.3; Panel C: Each serum from the PBS treated group was passed through an immunosorbent composed of rabbit anti-human IgG (open squares) or an immunosorbent composed of rabbit anti-mouse Ig (closed squares) prior to titration in the presence of
  • Figure 5 shows suppression of HAMA to the idiotype of the murine anti-DNP mAb, H1-DNP ⁇ -109.3, by Mab-2(mPEG) 36 conjugate.
  • Panel A and B hu-PBL-SCID mice injected with either 200 ⁇ g of Mab-2(mPEG) 36 (circles) or PBS (squares) were immunized with 5 ⁇ g of H1-DNP- ⁇ -109.3 in FCA 7 and 21 days later and bled 21 days after the second immunization (ie, 42 days after receipt of the tolerogen or PBS). All sera were diluted 1/100 in 0.01% normal murine Balb/c serum (NMS) and diluted two-fold in the presence of 0.01% NMS prior to being tested on DNP 9 -OVA coated plates. Each curve represents the data from the serum of one mouse.
  • NMS normal murine Balb/c serum
  • Panel A Each serum was titrated in the presence of a mixture of biotinylated and non-biotinylated Mab-2.
  • Panel B Each serum was titrated in the presence of a mixture of biotinylated and non-biotinylated H1-DNP ⁇ - 109.3.
  • Panel C Each serum from the PBS treated group was passed through an immunosorbent composed of rabbit anti-human IgG (open squares) or an immunosorbent composed of rabbit anti-mouse Ig (closed squares) prior to titration in the presence of biotinylated and non-biotinylated Mab-2.
  • Figure 6 shows suppression of HAMA to the idiotype of the murine anti-human CD4 mAb, Leu3a, by Mab-2(mPEG) 36 conjugate.
  • Each curve represents the data from the serum of one mouse.
  • the data depicted by the empty squares represent the proliferation of OVA-reactive T cells in the presence of OVA and in the absence of anti-CD4 mAb and of sera from hu-PBL-SCID mice.
  • the data depicted by the filled squares represent the background proliferation of OVA-reactive T cells in the absence of OVA, of anti-CD4 mAb and of sera from hu-PBL-SCID mice.
  • Panel A Titers obtained in the presence of OKT4 mAb for individual sera of control mice treated with PBS (in lieu of the tolerogen).
  • Panel B Titers of individual sera of mice treated with the tolerogen in the presence of OKT4.
  • Panel C Titers of individual sera of mice treated with PBS (in lieu of the tolerogen) in the presence of Leu3a;
  • Panel D Titers of individual sera of mice, which had been treated with the tolerogen, in the presence of Leu3a mAb.
  • Figure 7 shows a schematic illustration of the basic principles underlying the strategy for the protection of the donated heart from destruction by the patient's cellular and humoral immune responses.
  • Figure 8 is a flowchart of the strategy. The numbers refer to the procedures corresponding to the sections identified under "Specific Aims”. Median
  • SCID immunodeficiency
  • xenogeneic cells may be engrafted with a healthy individual's human peripheral blood leucocytes (hu-PBL).
  • hu-PBL peripheral blood leucocytes
  • very few of the SCID mice exhibit a transient graft versus host reaction.
  • the hu-PBL-SCID mice produce on immunization Ag-specific human Ab responses.
  • the present applicants have realised that the hu-PBL-SCID mouse system represents an ideal model for testing the immune responses of individual patients to a given BRM and the possibility of suppressing this response with tolerogenic conjugates.
  • the applicants used (as a model BRM) the murine mAb directed to ovalbumin (OVA), referred to as Mab-2, both as an immunogen and for the synthesis of the corresponding tolerogenic Mab-2(mPEG) 36 conjugate.
  • OVA ovalbumin
  • BALB/c scid/scid mice were obtained from Jackson Laboratories (Bar Harbour, ME) and bred by brother-sister mating under sterile conditions in the Central Animal Care Facility of the University of Manitoba.
  • a breeding nucleus of C.B-17 scid/scid mice was a generous gift of Dr. D.E. Mosier (La Jolla, CA). The sera of the
  • mice which had less than 50 ng/ml of mouse serum IgG were selected for breeding purposes and their Ig levels were monitored at 2- to 3-week intervals.
  • the Ig level of pregnant female mice were determined 2-3 days before delivery and that of their offspring at intervals of 3-4 weeks.
  • the mouse serum IgG levels were also monitored throughout the experiments at intervals of 3-4 weeks and animals having mouse serum Ig levels in excess of 500 ng/ml were eliminated.
  • PBL Peripheral Blood Leucocytes
  • erythrocytes were lysed by ammonium chloride.
  • the PBL of each donor were partitioned by passing through a nylon wool column into cell fractions enriched with respect to T cells (CD3 + > 90%; CD3-/DR + ⁇ 5%) and B plus MN cells (CD3 + ⁇ 10%;CD3-/DR + > 85% (Julius, M.H., et al. Eur. J. Immunol. 3, 645-9, 1973).
  • FCS heat inactivated fetal calf serum
  • DMSO fetal calf serum
  • the HLA type of each donor is given in the footnote to Table 3.
  • the PBL were freshly collected from a donor 12 months after immunization with diphtheria-pertussis-toxoid vaccine.
  • hybridoma cell line producing BALB/c anti-DNP mAb Hi-DNP-109.3; ⁇ 1 ⁇
  • Hybridoma cell lines producing mAbs to human CD3 (OKT3), human CD4 (OKT4), human CD8 (OKT8), K d (H-2K d 31-3-4S') and D d (H-2D d 34-4-20S) were purchased from
  • Biotinylated anti-HLA-DR mouse mAbs and avidin-FITC conjugates were obtained from Beckton Dickinson (Mountain View, CA).
  • the BALB/c anti-OVA mAb (Mab-2; ⁇ 1 ,i) was established in Winnipeg and purified from ascites as described (Lang, G.M. et al, 1992 supra).
  • Each of the other mAbs was purified from culture supernatants with the aid of protein A-agarose (Pierce, Rockford, IL) and/or of protein-G fast-flow (LKB-Pharmacia, Uppsala, Sweden) and the biotinylated mAbs were synthesized with biotin-LC-hydrazide (Piece, IL).
  • the OVA and purified protein derivative (PPD) were obtained, respectively from Sigma (St. Louse, MO) and CedarLane (Hornby, ON). Aggregate-free OVA was isolated by gel-filtration and was used as the immunizing Ag. To increase the immunogenicity of Mab-2, this mAb was heated twice at 63°C for 1 h; the resulting ha-Mab-2 was used as the immunizing Ag.
  • the purified, aggregate-free monomeric Mab-2 was converted to the tolerogenic derivative, Mab-2(mPEG) 36 , by reaction with the "activated intermediate" of mPEG
  • the tolerogenic fraction of the conjugate preparation was isolated from interfering contaminants (ie, high molecular weight cross-linked products, residual unmodified Mab-2, and unreacted but deactivated mPEG intermediate) by gel-filtration
  • ELISA plates (Corning Inc., Corning, NY) were coated with 100 ⁇ g/ml of rabbit anti-mouse Ig (Zymed, San Francisco, CA) or 10 ⁇ g/ml of rabbit anti-human Ig Abs which had ben absorbed with immobilized human or mouse serum Ig, respectively.
  • the ELISA plates were coated with Mab-2 (10 ⁇ g/ml). After coating, the plates were treated with 10% BSA for 2 h and, as usual, each serum, serially diluted, was added to the wells; the plates were maintained at 37°C for 4 h, or at 4°C overnight.
  • Protein A-purified serum IgG of BALB/c or of C57BL/6 served as standards for determination of murine Ig in sera of BALB/c or C.B-17 scid/scid mice respectively.
  • Human IgG isolated by gel filtration was used as a standard for the determination of the human Ig in sera of SCID mice. After incubation, the ELISA plates were washed with PBS containing 0.01% Tween 20;
  • biotinylated rabbit anti-mouse Ig Abs Zymed, South San Francisco, CA
  • biotinylated mouse anti-human Ig mAb Zymed; HP
  • the plates were incubated at 37°C for 2 h or at 4oC overnight, washed 4 times, and finally the amounts of biotinylated Abs were determined colorimetrically at 405 nm by reaction with alkaline phosphatase-strepavidin conjugates (Zymed), and nitrophenyl phosphate (Sigma) as the substrate.
  • mice Twelve ours after engraftment of PBL, the test and control hu-PBL-SCID mice received an i.p. injection of 200 ⁇ g of Mab-2(mPEG) 36 or PBS, respectively. Seven and 21 days later, ie on days 8 and 28, all mice received two i.p. immunizing injections of 20 ⁇ g of ha-Mab-2 or 100 ⁇ g of OVA. On day 42 the serum of each mouse was assayed by ELISA for total human Ig Abs to Mab-2, and for total human IgG levels.
  • human T cells were isolated from spleen and lymph nodes of each hu-PBL-SCID mouse by passage through nylon wool column.
  • the non-adherent cells were treated with a mixture of anti-H-2- d mAbs (ie, anti-K d and anti-D d mAbs), or with a mixture of anti-H-2 d mAbs and 0KT4, or a mixture of anti-H-2 d mAbs and OKT8, in the presence of rabbit complement (RC).
  • the protocol for this test is illustrated in the flowchart in Figure 3.
  • the culture medium was RPMI-1640, containing 5% human cord serum and 5 ⁇ 10 -5 M of 2-mercaptoethanol.
  • SCID mice were engrafted with 50 ⁇ 10 6 PBL of each of the three volunteers and immunized s.c. with 100 ⁇ g of Mab-2 in Freund's complete adjuvant (FCA).
  • FCA Freund's complete adjuvant
  • the spleen and lymph node cells of each mouse were isolated and pooled, and treated with a mixture of anti- H-2 d mAbs plus RC'.
  • the viable cells were stimulated twice with 100 ⁇ g/ml of Mab-2 or 20 ⁇ g/ml of PPD at 21 days intervals. Two weeks after the last stimulation, the cultured cells were harvested and then treated with OKT8 plus RC. The residual cells were used as responder CD4 + T cells.
  • each SCID mouse was engrafted with 50 ⁇ 10 6 PBL of volunteer "A” and 12 hr later they were injected i.p. with 200 ⁇ g of Mab-2(mPEG) 36 or PBS, and all the mice received i.p. an immunizing
  • CD8 + T cells were isolated from the pooled spleen and lymph node cells of each hu-PBL-SCID mouse by passage through nylon wool column, and treated with a mixture of anti-H-2 d and OKT4 mAbs in the presence of RC'.
  • irradiated hu-PBL served as the source of APC.
  • the cells were pulsed for the last 12 h of culture with 3 H-TdR
  • Mab-2 ie, ha-Mab-2, or of Mab-2 emulsified in FCA on days 0 and 28, even though more than 1.0 mg/ml of total human Ig was detected in some of the mice (data not shown). Since B cells represent only 15-20% of PBL, it was decided to transfer into the SCID mice a cell
  • HAMA responses were compared with the human Ig levels in sera of both control and test hu-PBL-SCID mice, ie, mice which had received, respectively, PBS and Mab-2(mPEG) 36 prior to immunization.
  • total human IgG levels of all hu-PBL-SCID mice on day 42 after engraftment with 20 ⁇ 10 6 T and 20 ⁇ 10 6 (B+MN) cells of a single donor (ie, volunteer "A”) were in the range of 3-5 mg/ml.
  • no obvious differences in total human IgG levels were observed on comparing these levels for control and tolerized hu-PBL-SCID mice.
  • the induced HAMA responses varied among the animals within each group; nevertheless, the results in Table 1
  • responder T cells of each donor showed significant proliferative activity by stimulation with the relevant Ag in the absence or presence of CD8 + T cells of hu-PBL-SCID mice which had not been treated with the conjugate.
  • CD8 + T cells of tolerized hu-PBL-SCID mice which had been engrafted with hu-PBL of donor "A" caused significant reduction of proliferation
  • CD8 + T cells of donor "B” who shared class I Ags, ie, A2, A24, B51, c1, c3, and Class II ags, ie, DR4, which those of donor "A”.
  • CD8 + T cells "A” did not decrease the response of T cells of donor "C” against Mab-2 and PPD, whose class I Ags, c1, and class II Ags, DR4 and DR9, were identical with those of volunteer "A”.
  • TT anti-tetanus toxoid
  • the failure of detection of HAMA responses in hu-PBL-SCID mice may be due to a low number of B cells committed to mouse Ig in a given batch to hu-PBL rather than to a limited clonal heterogeneity of the B cells, which had survived in the mice (Saxon, A., et al, J. Clin. Invest. 87, 658- 665, 1991). More recently, it was reported that human mature T cells in hu-PBL-SCID mice were refractory to simulation by anti-CD3 mAb, but they proliferated in response to exogenous 11-2 (Tary-Lehmann, M., et al, J. Exp. Med.
  • SCID mice Severe combined immunodeficient mice were reconstituted with normal human peripheral blood
  • leucocytes PBL and were shown to produce a human anti-mouse Ig antibody (HAMA) response on immunization with heat-treated murine monoclonal IgG1 antibody (mAb) to ovalbumin (OVA), referred to a ha-Mab-2.
  • HAMA human anti-mouse Ig antibody
  • mAb murine monoclonal IgG1 antibody
  • OVA ovalbumin
  • the HAMA response was proportional to the number of B cells and mononuclear cells transferred from a given batch of PBL.
  • pretreatment of hu-PBL-SCID mice pretreatment of hu-PBL-SCID mice with a
  • mice Groups of four, 3-4 week old, BALB/c SCID mice received i.p., on day 0, the indicated numbers of T and (B+MN) cells, which had been isolated from the PBL of a healthy volunteer "A" by passage through a nylon wool column. The freshly isolated cells served for Experiment 1. For Experiment 2, cells which had been maintained in liquid nitrogen were used. Twelve hours after cell transfer, all test mice received i.p. 200 ⁇ g of Mab- 2(mPEG) 35 and the control mice received PBS instead. All mice received two immunizing injections of either 20 ⁇ g of ha-Mab-2 of 100 ⁇ g of aggregate-free OVA on days 8 and 21. The mice were bled on day 35 and their sera were assayed by ELISA for human IgG antibodies to Mab-2 and OVA.
  • mice received i.p. on day 0 the indicated number of T and (B+MN) cells of two healthy volunteers "B" and "C", which had been kept in liquid nitrogen. Twelve hours later, the test mice received i.p. 200 ⁇ g of Mab-2(mPEG) 36 and the control mice received PBS. All mice were immunized with 20 ⁇ g of ha-Mab-2 on days 8 and 21. On day 35, the ELISA titers of human IgG antibodies to Mab-2 were individually determined. Table 2 INDUCTION OF CD8 + Ts CELLS IN HU-PBL-SCID MICE BY Mab-2(mPEG) 36 *
  • CD4 + T cells a,b added No addition Addition of CD8 + T cells of of of cells "primeid mice” "tolerized mice”
  • mice were similar to those developed by Mosier D.E. et al in Nature 335, 256-259, 1988, J. Clin. Immunol. 10, 185-191, 1990; Science 251, 791-794, 1991.
  • BALB/c scid/scid and C.B-17 scid/scid mice were used.
  • Each mouse was engrafted with 20 ⁇ 10 6 T cells and 20 ⁇ 10 6 of a mixture of B and mononuclear (B+MN) cells, which had been isolated by leukaphoresis from the blood of one healthy, EBV-, HIV- and HBV-negative
  • the test mice used for the Mab-2 and H 1 systems received 12 h after engraftment of the human cells a single i.v. injection of 200 ⁇ g of the immunosuppressive Mab-2(mPEG) 36 and the control mice were administered PBS.
  • the mice which were used for the Leu-3a system received 100 ⁇ g of Mab-2(mPEG) 36 .
  • This serum will be referred to as
  • the CD4 + T cells of the same donor (whose hu-PBL cells had served for engraftment of the SCID mice) were used.
  • these hu-PBL cells were first inactivated by mitomycin C (MMC) and 10 ⁇ 10 7 of these cells were incubated with 1 ⁇ g of Leu3a for 45 min at 0°C and washed with RPMI-1640 medium, and the Leu-3a coated CD4 + T cells were then used for immunization.
  • MMC mitomycin C
  • Leu-3a coated CD4 + T cells were then used for immunization.
  • these cells were maintained in 25 ⁇ g of MMC/ml for 60 min at 37°C.
  • the CD4 + T cells were prepared by stimulating 10 ⁇ 10 6 of the hu-PBL in culture with 100 ⁇ g/ml of OVA at 2-3 week intervals. Ten days after the third stimulation the cultured cells were harvested, passed through a Sephadex G-10 column, treated with OKT8 in the presence of rabbit complement (RC), and washed thrice with RPMI-1640 medium and centrifuged; the viable cells were used as the OVA-sensitive CD4 + T cells.
  • each serum of the control and test hu-PBL-SCID mice was diluted 10-fold with RPMI-1640 medium containing 5% of human cord serum as a source of lymphokines, 5 ⁇ 10 -5 M of 2-mercaptoethanol, and 10 ng/ml of protein A-purified mouse IgG, and
  • the OVA-reactive CD4 + T cells (5 ⁇ 10 5 ) were co-cultured for 2 days with 1 ⁇ 10 5 irradiated (2000 rad) autologous (B+MN) cells, as a source of APC, in the presence of OVA (100 ⁇ g/ml), and of Leu3a (10 ng/ml) and of each serum at different dilutions. Finally, the cells were pulsed with 3 H-TdR (0.5 ⁇ Ci/well) and the culture was continued for 12 additional hours.
  • Balb/c SCID mice received two s.c. injections of 1 ⁇ g of Mab-2 in Freund's complete adjuvant (FCA) on days 7 and 28 after administration of Mab-2(mPEG) 36 or PBS. On day 42 all mice were bled for determination of their anti-id Abs.
  • FCA Freund's complete adjuvant
  • determinants of the Ig eg, isotypic determinants
  • mPEG conjugate which is responsible for induction of the Ts cells.
  • mAbs ie, xenogeneic, chimeric, humanized, or even "human” mAbs produced by genetic engineering
  • tissue antigens either by themselves, or as immunoconjugates with toxins
  • a truly subclass pan-specific tolerogenic mPEG conjugate of a murine mAb capable of suppressing the HAMA responses to the different idiotypic epitopes of mAbs directed to diverse antigens.
  • it will be essential either (i) to incorporate the epitopes specifying the different isotypic subclass determinants of the light and heavy chains into the engineered Ig onto which will be grated the requisite number of mPEG molecules, or (ii) to use different mAbs possessing the same heavy and light chains as the mAb used for the synthesis of the corresponding mPEG conjugate.
  • SCID mice Severe combined immunodeficient mice were reconstituted with normal human peripheral blood
  • hu-PBL leucocytes
  • mPEG monomethoxypolyethylene glycol
  • mAb anti-ovalbumin, IgGl murine monoclonal antibody
  • Mab-2 suppressed the HAMA responses to both the common ( ⁇ 1, ⁇ ) and the idiotypic determinants of Mab-2.
  • the Mab-2(mPEG) 36 conjugate suppressed the immune responses of hu-PBL-SCID mice to the common and idiotypic determinants of murine mAbs to the DNP residue and to human CD4, which mAbs consisted also of ⁇ 1 and ⁇ chains.
  • the ISDs are often administered in conjunction with mAbs to specific determinants of human T cells, e.g., CD3, CD4 and the IL-2 receptor; these mAbs lead to the downregulation of T-dependent immune responses.
  • mAbs to specific determinants of human T cells e.g., CD3, CD4 and the IL-2 receptor; these mAbs lead to the downregulation of T-dependent immune responses.
  • the use of ISDs is limited by their side effects.
  • heart graft survival depends on the degree of cross-matching of the
  • HLA human leucocyte antigen
  • HLAs g 1 to g 4
  • g 2 the incompatible HLA recognized uniquely by the patient's Th 2 and/or Tc 2 cells.
  • the cytotoxic T cell CTL or TC
  • the Th 2 cell is essential for activating the patient's Tc 2 cells. Therefore, administration of the two corresponding Abs ( H T- H T, ), which recognize the unique antigenic determinants of V ⁇ 2 /V ⁇ 2 and V ⁇ 2 '/V ⁇ 2 ' of the TCRs of the respective Th 2 and Tc 2 cells, should annul the participation of these T cells in the rejection
  • a-cAbs anti-clonotypic Abs
  • Th helper T cells
  • TCR Tc cells
  • the applicant proposes the application of the method of pegylation of protein Ags to the suppression of HAMA responses in prospective heart transplant recipients.
  • Step I Injection of the tolerogenic conjugate, Ag(mPEG) n , leading to the generation of Ag-specific suppressor T (Ts) cells.
  • Step II Administration of the unmodified Ag at least 7 days after Step I. Thereafter, the Ag can be injected repeatedly over extended periods without further injections of Ag(mPEG) n .
  • This two-step procedure rather than injection of only the tolerogenic mPEG conjugate, is due to the fact that coupling of the required number of the long chains of mPEG onto a protein for converting it to a tolerogen (i.e., 10 ⁇ 2 mPEG molecules per protein unit of about 50 KDa), results in the masking of some of the epitopes and in structural modifications of the original Ag and, consequently, in loss of its biological and ligand binding activities.
  • a tolerogen i.e. 10 ⁇ 2 mPEG molecules per protein unit of about 50 KDa
  • the applicant proposes using the immunosuppressive method based on tolerogens for the selective elimination, or radical downregulation, of the T cells of the recipient of the graft which recognize the allo-Ags of the donor's heart and are responsible for its rejection.
  • transplants in man can be achieved by generating anti-clonotypic mAbs (a-cmAbs) in mice or rats which will recognize the clonotypic Ags of the T cell receptors (TCRs) of the patient's Th and Tc cells.
  • a-cmAbs anti-clonotypic mAbs
  • TCRs T cell receptors
  • a-cmAbs should react with the unique V ⁇ /V ⁇ determinants of these cells and, thus, either inactivate these T cells and/or block their interaction with the allograft.
  • TCRs T cell receptors
  • this strategy may involve the following 2 phases:
  • Phase II - This phase consists of (a) injection, still under the umbrella of ISDs, of the tolerogenic mPEG conjugates of the above a-cmAbs for activating the Ts cells which will suppress the immune response of the patient specifically to the a-cmAbs, and (b)
  • the immunological status of the patient will be monitored at varying intervals with respect to the possible re-appearance of the CTLs, which are expected to have been ablated by the a-cmAbs.
  • This strategy should have advantages over the current blunderbuss approach, involving the indiscriminate use of ISDs and of mAbs to the patient's T cell determinants, which leads to
  • the mPEG conjugates may be synthesized according to methods as previously described. Necessary immunological procedures are commonly used in many laboratories.
  • H-2K bm molecules differ from the H-2K molecules by 2-5 amino acid substitutions which reflect the corresponding gene substitutions, and (iv) the various K b mutations alter the K b molecule sufficiently so that they differ antigenically.
  • the rates of rejection of skin grafts from female B6 bm mice by female B6 mice followed a distinct hierarchy: bm1 skin
  • the overall strategy incorporates the following aims.
  • tissue allografts may be due to different B6 anti-K bm3 reactive cells generated in
  • Th cells of the B6 recipient specific to the graft's allo-Ags may be
  • B6 Tc cells will be generated in the draining lymph nodes of the recipient. These Th and/or Tc cells would then be generated in the draining lymph nodes of the recipient.
  • Th and Tc cell lines will be
  • the grafted hearts are treated with collagenase (1mg/ml) and DNase (80-100 ⁇ g/ml for 1 h at 37°C); the Thy-1 + T cells are isolated from the cell suspension with magnetic
  • the isolated T cells are cultured by stimulation at 2-week intervals with irradiated (3,300 rad) spleen cells of bm3 mice in the presence or absence of con A-sup (supernatants from rat spleen cells stimulated for 40 hr with 5 ⁇ g/ml of concanavalin A as source of T-cell growth factors) according to the method established by Kimoto and Fathman (1980 J. Exp. Med. 152, 759-770) with slight
  • the cells of the draining lymph nodes are also to be cultured by the use of this method. After the 3rd or 4th stimulation, single cells are to be picked out from the culture by micro-manipulation for the generation of the corresponding B6 anti-bm3 Th cloned cell lines.
  • class I Ags was shown to be initiated by CD8 + and CD4 + Th cells, which recognize class I allo-Ags on allogeneic Ag presenting cells (APC), or in
  • the generated Th cell clones may be selected on the basis of their ability to secrete IL-2.
  • the following two batches of stimulator cells are: (i) one batch of cells prepared from spleen cells of bm3 mice by irradiation at 3,300 rad (these cells will be referred to as APC + stimulator cells), and (ii) another batch of cells isolated by passage of the irradiated spleen cells through Sephadex G-10 columns (Ly, I.A. et al., 1974 J. Immunol. Meth. 5, 239-247) to remove the adherent APC (these cells will be referred to as APC- stimulator).
  • Th cell lines To obtain cloned Th cell lines, they are to be cultured with APC + or APC- stimulator cells in the
  • anti-IL-2R mAbs 7D4 (Malek, T.R. et al., 1983 P.N.A.S. 80, 5694-5698, and in the absence or presence of the irradiated B6 spleen cells (as source of self APC).
  • the reason for the addition of anti-IL-2R mAbs to each culture is to minimize, if not completely inhibit, the consumption of IL-2 produced by allo-Ag reactive T cells by the other cells in the culture; this procedure results in increased sensitivity of the assay.
  • the IL-2 content of the culture supernatants may be determined by a standard method utilizing IL-2
  • the clones generated are from (i) T cells of draining lymph nodes, and (ii) T cells
  • the T cells infiltrating the graft and residing in the draining lymph node may be maintained by an established procedure
  • the cells from these two sources are stimulated with irradiated bm3 spleen cells at intervals of 5-7 days.
  • the cultured cells are diluted and maintained in 24-well culture plates (1 ⁇ 10 5 /well) with RPMI-1640 medium
  • the generated clones may be selected on the basis of their ability to lyse 51 Cr-labelled LPS- and con A-blast cells of bm3 spleen cells.
  • the CTL clones may also generated from several batches of grafted hearts and lymph nodes of different mice.
  • the surface phenotypes of each T cell clone may be determined by fluorometric analysis with a flow cell sorter (EPICS model 753, Coulter, CA).
  • a flow cell sorter EPICS model 753, Coulter, CA.
  • the cells are first treated with biotinylated mAbs specific to Thy-1.2 (HO.13.4), CD4 (YST191.1), CD8
  • V ⁇ /V ⁇ genes of the TCRs of T cells infiltrating allogeneic hearts, implanted in B6 mice between their kidneys and kidney capsules, at different stages of rejection.
  • the V ⁇ /V ⁇ genes of the cloned T cell lines correspond to the T cells which actually infiltrate the transplanted heart, since one may visualize that the T cells selected for cloning may represent cells proliferating preferentially under the culture conditions.
  • a piece of adult bm3 heart is to be implanted between the kidney and the capsule of each B6 mouse (bm3 mice will serve for controls).
  • the infiltrated T cells are to be isolated, as described above, and the CD4 + and/or CD8 + T cells separated from the isolated T cells with Dynabeads coated with anti-CD4 mAb or anti-CD8 mAb, respectively.
  • Total RNA may be extracted from these cells by the standard guanidium thiocyanate-cesium chloride method (Chirgwin, J. M. et al., 1979 Biochem. 18, 5294-5299) and the first strand of cDNA synthesized using 5 ⁇ g of total RNA in a 20 ⁇ l reaction mixture consisting of oligo(dT) primer and AMV reverse transcriptase. Both the V ⁇ and V ⁇ genes may be amplified by the polymerase chain reaction (PCR) utilizing the Taq polymerase according to the method of Maeda et al . (1991, Diabetes 40, 1580-1585).
  • PCR polymerase chain reaction
  • each of the seventeen different V ⁇ primers may be used in
  • V ⁇ and C ⁇ primers may serve as controls.
  • the synthetic oligonucleotides of all V ⁇ primers and C ⁇ primers may be purchased from the DNA Synthesis Laboratory, University of Manitoba.
  • the amplified PCR products may be hybridized with 32 P-labelled HinfI-EcoRI fragment of C ⁇ (86T5) gene (Hedrick, S.M. et al., 1984 Nature 308, 153-158).
  • the intensities of the auto-radiographic bands may be quantified by densitometry.
  • V ⁇ gene frequencies may also be determined by this procedure utilizing the corresponding V ⁇ , 3'-C ⁇ and 5'-C ⁇ primers.
  • CD4 + and CD8 + Th cells initiate the graft reaction, and if CD8 + and CD4 + CTLs play the major role as effector cells in the rejection episode.
  • V ⁇ /V ⁇ genes of each T cell clone Each ss DNA may be hybridized with cloned gene fragments of 15 different V ⁇ and 11 different V ⁇ genes (kindly provide/d by Dr. E. Palmer, Denver, CO). After the determination of the family of V ⁇ and V ⁇ genes, the V ⁇ and V ⁇ cDNAs may be amplified utilizing the corresponding V ⁇ primers and 3'-C ⁇ , and the corresponding V ⁇ primers and 3'-C ⁇ , in order to determine the J ⁇ and D ⁇ -J ⁇ gene usages. Each amplified product will be cloned into pCRlOOO vector (In Vitrogen, CA) and will be
  • rat a-cmAbs are to be generated to the V ⁇ -J ⁇ / /V ⁇ -D ⁇ -J ⁇ regions of the TCRs of the CD4 + and CD8 + T cells involved in the early phase; these a-cmAbs should inhibit the initiation of graft rejection.
  • rat a-cmAbs are to be produced against the V ⁇ /V ⁇ regions of the CD8 + and CD4 + CTL clones, derived from T cells infiltrating the heart graft during the middle phase of the episode; these a-cmAbs should inhibit the continuing destruction of the transplanted heart.
  • Lewis rats are immunized twice with each of the identified Th and CTL clones at an interval of 2 weeks. Seven days after the second immunization, all rats are bled and each of their sera absorbed with B6 spleen cells to remove antibodies to common murine Ags. After absorption each serum is tested for its capacity to inhibit (i) the proliferation of the corresponding Th cell clones in the presence of allo-Ags, or (ii) the cytolysis of LPS blast cells of bm3 mice by the
  • the rats producing the desired a-cAbs are immunized once more and 2 days later their spleen cells fused with mouse myeloma cells,
  • the IgG a-cmAbs to Th and CTL clones may be screened by the inhibition of proliferation of the corresponding Th cell lines, or by the suppression of cytotoxic
  • the selected mAbs may be subjected to further selection on the basis of their capacity to inhibit the MLR between polyclonal B6 spleen cells and bm3 cells, or the cytotoxicity of polyclonal B6 CTLs using 51 Cr-labelled LPS blast cells of bm3 mice.
  • mAbs-8H or mAbs-4H The mAbs directed against the clonotypes of the B6 CD8 + and CD4 + Th cells will be referred to as mAbs-8H or mAbs-4H, respectively, and the corresponding mAbs to the clonotypes of the CTLs recognizing the epitopes of the bm3 mutant will be referred to as mAbs-K.
  • rat IgG mAbs to V ⁇ /V ⁇ regions of cloned Th cells should suppress the MLR between host and donor cells and are, therefore, to be used to explore their efficacy in "immunoprophylaxis" by inhibition of immune responses of the graft recipient, which lead to the graft's rejection.
  • a Regimen for the immune prophylaxis is proposed, consisting of two steps utilizing for the exploratory experiments using skin grafts.
  • Step I The B6 mice receive the tolerogenic mAbs- 8H(mPEG) n and/or mAbs-4H(mPEG) n , for induction of
  • Step II Beginning 7 days later, the B6 mice receive i.v. injections of the "prophylactic mAbs", at intervals determined by the half-life of these mAbs. On the eighth day pieces of skin of bm3 mice are grafted onto the tail of B6 mice for the determination of the efficacy of this therapy.
  • the control mice may reject the allograft within 14 to 30 days (as stated earlier, the MST depends on the bm strain combinations). A marked prolongation of MST, will lead to testing of this therapy for heart transplantation, i.e., hearts will be grafted heterotopically in the abdominal cavity by the method of Corry et al. (1973 Transplantation 16, 343-350).
  • rat mAbs to B6 anti-bm3 CD8 + CTL clone(s), [i.e., to mAbs-K(s)] are to be used for the "rescue therapy", i.e., these mAbs will be administered to B6 mice undergoing rejection of bm3 skin grafts. Since, in contrast to the "prophylaxis therapy", this clinical intervention has to be applied without any delay, the arrest of the ongoing graft rejection episode may require the injection of mAbs-K prior to tolerization of the host by
  • the first therapeutic modality involves the i.v. injections of mAbs-K at intervals of 2-4 days without ISD. If prolongation of MST is achieved by this procedure, the same regimen is to be tested for heart transplantation by the method of Corry et al. (1973 supra).
  • the grafting of the skin is to be done under the umbrella of ISD with simultaneous
  • the proposed therapeutic strategy could be simplified.
  • the a-cmAbs in a two-step procedure, in conjunction with their tolerogenic mPEG conjugates, one may use directly the mPEG conjugates of the Ag-binding fragments of the a-cmAbs, i.e., the Fab', or the (Fab') 2 , or the Fav, or even the antigen binding region of the H chains of these a-cmAbs.
  • the present invention encompasses methods of
  • compositions for use in methods of treatment compositions for use in methods of treatment, the use of compositions in treatment, the use of
  • compositions in the manufacture of medicaments for use in treatment and products containing compositions as a combined preparation for simultaneous, separate or sequential use in therapy; as described herein.

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Abstract

Cette invention se rapporte à un procédé de vérification de la réponse immune individuelle d'un patient (P) à un anticorps 'X' ou à un fragment de liaison de celui-ci, qui est spécifique à un épitope d'un médiateur de la réponse immune du patient contre l'antigène 'Z'. Ce procédé consiste à : (i) isoler une échantillon des leucocytes du sang périphérique (PBL) du patient, (ii) greffer une souris présentant un déficit immunitaire combiné sévère (SCID) avec ledit échantillon afin de créer une souris-test p-PBL-SCID présentant les PBL du patient, (iii) traiter ladite souris-test avec un tolérogène conçu pour être administré au patient afin de réduire la réponse immune de ce dernier à l'anticorps 'X', (iv) après l'étape (iii), immuniser la souris à l'aide de l'anticorps 'X': (v) après les étapes (iii) et (iv), détecter toute réponse d'anticorps humain chez la souris. Le tolérogène comprend un conjugué covalent soluble dans l'eau d'un anticorps 'Y' avec un ou plusieurs polymères non-immunogènes solubles dans l'eau, l'anticorps 'Y', bien que différent de l'anticorps 'X', présente une structure commune avec ce dernier.
PCT/GB1993/001903 1992-09-09 1993-09-09 Procede de verification de reponse immune individuelle a un anticorps a l'aide d'une souris a scid WO1994005779A1 (fr)

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AU49770/93A AU4977093A (en) 1992-09-09 1993-09-09 Testing individual immune response to an antibody with scid mouse
EP94908820A EP0662127A1 (fr) 1992-09-09 1993-09-09 Procede de verification de reponse immune individuelle a un anticorps a l'aide d'une souris a scid

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GB9219063.6 1992-09-09
GB929219063A GB9219063D0 (en) 1992-09-09 1992-09-09 Suppression of allograft rejection
GB9226009.0 1992-12-14
GB929226009A GB9226009D0 (en) 1992-12-14 1992-12-14 Immunosuppression

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP2422618A1 (fr) 2010-08-27 2012-02-29 Technologie Integrale Ltd. Modèle animal pour l'évaluation de l'efficacité d'un vaccin contre le VIH

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EP0438053A1 (fr) * 1990-01-15 1991-07-24 Yeda Research And Development Company Limited Implantation durable et développement d'une lignée hématopoiétique humaine chez des mammifères normaux
EP0469632A1 (fr) * 1990-08-03 1992-02-05 Systemix, Inc. Systèmes des organes xénogènes modulés dans un hôte immunocompris
EP0517199A1 (fr) * 1991-06-04 1992-12-09 Yeda Research And Development Company, Ltd. Transplantation durable de cellules et de tissu humain dans des mammifères normaux
WO1993005796A1 (fr) * 1991-09-19 1993-04-01 The Scripps Research Institute Procede de production d'anticorps humains dans un animal non humain, et animaux utilises a cet effet
WO1993012252A1 (fr) * 1991-12-18 1993-06-24 Nederlandse Organisatie Voor Toegepastnatuurwetenschappelijk Onderzoek Tno Procede de mise a l'epreuve d'agents antiviraux et animaux d'experience utilises dans ce procede

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EP0438053A1 (fr) * 1990-01-15 1991-07-24 Yeda Research And Development Company Limited Implantation durable et développement d'une lignée hématopoiétique humaine chez des mammifères normaux
EP0469632A1 (fr) * 1990-08-03 1992-02-05 Systemix, Inc. Systèmes des organes xénogènes modulés dans un hôte immunocompris
EP0517199A1 (fr) * 1991-06-04 1992-12-09 Yeda Research And Development Company, Ltd. Transplantation durable de cellules et de tissu humain dans des mammifères normaux
WO1993005796A1 (fr) * 1991-09-19 1993-04-01 The Scripps Research Institute Procede de production d'anticorps humains dans un animal non humain, et animaux utilises a cet effet
WO1993012252A1 (fr) * 1991-12-18 1993-06-24 Nederlandse Organisatie Voor Toegepastnatuurwetenschappelijk Onderzoek Tno Procede de mise a l'epreuve d'agents antiviraux et animaux d'experience utilises dans ce procede

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BITOH, S. ET AL.: "Specific suppression of human antimurine antibody hama responses including anti-idiotypic responses in hu-PBL-SCID mice", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 91, no. 1PT2, January 1993 (1993-01-01), pages 142 *
BITOH, S. ET AL.: "Suppression of human antibody response in hu-PBL-SCID mice by tolerogenic conjugates of antigen and monomethoxypolyethylene glycol MPEG", FASEB JOURNAL, vol. 6, no. 5, 5 April 1992 (1992-04-05), BETHESDA, MD US, pages A2008 *
BITOH, S. ET AL.: "Suppression of human anti-mouse idiotypic antibody responses in hu-PBL-SCID mice", HUMAN ANTIBODIES AND HYBRIDOMAS, vol. 4, no. 3, July 1993 (1993-07-01), pages 144 - 151 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2422618A1 (fr) 2010-08-27 2012-02-29 Technologie Integrale Ltd. Modèle animal pour l'évaluation de l'efficacité d'un vaccin contre le VIH
WO2012025167A1 (fr) 2010-08-27 2012-03-01 Technologie Integrale Ltd. Modèle animal pour l'évaluation de l'efficacité d'un vaccin contre le vih

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