AU615233B2 - Monoclonal antibody that recognizes an epitope of the gamma chain of human t cell surface receptors - Google Patents

Description

I 1

PCT

WORLD INTELALECTU-AI R y ORGANIZATION International Bureau 0 INTERNATIONAL APPLICATION PUBLISHED UNI P N P N IO ATXN TREATY (PCT) (51) International Patent Classification 4 (ll) ernaionaWubl~ onWnbe WO 89/ 02899 C07K 15/04, C12N 5/00 A l (43) International Publication Date: 6 April 1989 (06.04.89) (21) International Application Number: PCT/US88/03123 (81) Designated States: AT (European patent), AU, BE (European patent), BR, CH (European patent), DE (Eu- (22) International Filing Date: 9 September 1988 (09.39.88) ropean patent), DK, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), NO, SE (31) Priority Application Number: 87/13532 (European patent).

(32) Priority Date: 30 September 1987 (30.09.87) Published (33) Priority Country: FR With international search report, BeJbre the expiration of the time limit for amending the claims and to be republished in the event of the receipt (71) Applicant: COULTER CORPORATION [US/US]; 600 f amendments.

West 20th Street, Hialeah, FL 33010 (US).

(72) Inventor: HERCEND, Thierry 39, rue Camille-Desmoulins, F-94805 Villejuif C6dex (FR).

(74) Agents: CASS, Myron, C. et al.; Silverman, Cass, Sing- JU 1989 er Winburn, Ltd., 105 West Adams Street, 27th Floor, Chicago, IL 60603 AUSTRALIAN 18 APR 1989 PATENT OFFICE (54) Title: MONOCLONAL ANTIBODY THAT RECOGNIZES AN EPITOPE OF THE GAMMA CHAIN OF HU- MAN T CELL SURFACE RECEPTORS (57) Abstract The present invention relates to a novel monoclonal antibody of the anti-TigammaA type. This monoclonal antibody recognizes a unique receptor of the T lymphocytes of the "second family" encoded by genes called "gamma" which rearrange in T cells specifically, The invention can be used in the diagnosis and the treatment of auto-immune diseases, carcinoma and viral infections, for instance.

i .WO 89/02899 PC /US88/03123 -1- Description MONOCLONAL ANTIBODY THAT RECOGNIZES AN EPITOPE OF THE GAMMA CHAIN OF HUMAN T CELL SURFACE RECEPTORS Technical Field This invention relates generally to monoclonal antibodies and their interaction with specific antigenic determinants, and relates to a hybridoma celi line which produces a monoclonal antibody which specifically binds to the cell surface receptor of human T lymphocytes. More particularly, the monoclonal antibody embodying the invention binds a unique epitope of the human T cell surface receptor encoded by a T cell gamma rearranging gene.

Background Art In this technical field, it is well known that the human immune system is comprised of cellular and soluble elements in blood together with various organs of the lymphoreticular system. A foreign substance, called an antigen, entering the body system, when identified, will result in an immune system response or reaction. The reaction can take the form of a localized inflammation or phagocytic reaction or may result in a specific humoral or cell mediated reaction. These specific reactions require the interaction of lymphocytes to produce antibody against the foreign substance, which may be a cell, virus or bacteria, for instance. The major elements in an immune response are T cells derived from the thymus and B cells derived from bone marrow. The T cells will interact with the B cells to create an antibody formation against the foreign substance in what is termed a cell mediated immunity. The humoral immune response requires only the presence of a presensitized B lymphocyte together with a foreign substance. Their interaction causes the B cells to proliferate and begin synthesizing immunoglobulins or antibody protein against the antigen.

Circulatory peripheral blood cellular components com- WO 89/02899 PCT/US88/03123 -2prise red blood cells or erythrocytes, white blood cells or leukocytes and platelets. The leukocytes are divided into five major populations called neutrophils, eosinophils, basophils, monocytes and lymphocytes. The lymphocytes are further subdivided into thymt. derived T cells and bone marrow derived B cells. The T and B lymphocytes are further divided into numerous subsets, such as inducer cells, suppressor cells, killer cells, among others, with increasing numbers of subsets being identified in current research efforts.

The monoclonal antibody technology derived from the work of Koehler and Milstein in 1975, Nature, 256, 495-497 has lent great impetus to scientific research into the functions of leukocytes and their human physiology implication. Monoclonal antibodies were developed to human T lymphocyte and B lymphocyte surface antigens or epitopes in their various stages of development and maturation and to classify B and T cell malignancies, such as T and B cells leukemias and lymphomas. The availability of monoclonal antibodies which recognize various functionally rslevant T or B cell surface molecular structures allows a.detailed characterization of both the process of T cell activation and the distinct subsets of T cells.

Further, monoclonal antibodies have been developed which bind solid tumor cells, glycoprotein surface molecules of red blood cells, and inflammatory sites of human tissues infected by virus, such as in AIDS diseases, just to name a few. The effective study of human cells, both normal and abnormal, for their function and impact on the human immune system increases spectacularly as new monoclonal antibodies are developed for diagnostic and tberapeutic applications, both in vitro and in vivo.

In United States Letters Patent No. 4,550,086 titled "Monoclonal Antibodies That Recognize Human T Cells", attention is directed to definition of surface molecules expressed on the T lymphocytes and other cells, such as structures designated: "T 4 and WO 89/02899 PCT/US88/03123 -3- The monoclonal antibody designated "Anti-T 3 to T3" was able to block the ability of any functional T cell clone to recognize its target antigen. It was learned that each different mature T cell clone has a unique protein molecular structure which enables each T cell to recognize its target antigen. Hovrawer, the patent teaches that although T cell clones may differ in their so-called "recognition structure", they appear to have certain common characteristics.

One common characteristic of all T cell clones is constituted by the presence of T 3 glycoproteins and of a dimer composed of two different protein molecules as identified in the said patent. The monoclonal antibody described in the patent recognized specifically the T cell surface receptor complex.

The monoclonal antibody embodying the invention recognizes a sub-population of human peripheral T cells which represents approximately 1-15% of circulating mononuclear blood cells and a mean value of about 3% of these lymphocytes. The lymphocytes of this subpopulation express a T cell surface receptor of the "second family", encoded by genes of the gamma class which rearrange specifically in the T cells. The monoclonal antibody of the invention binds to a unique epitope of a glycoprotein molecule on the surface of human T lymphocytes having a molecular mass of about 85,000 daltons determined under non-reducing conditions and of 41,000-44,000 daltons determined under reducing conditions. The unique epitope characterized by this monoclonal antibody enables characterization of a sub- K! population of human T lymphocytes with greater improved definity than heretofore achieved.

The nmonoclonal antibody of the invention identifies a novel sub-set of human lymphocytes with T cell receptor gamma complex. The monoclonal antibody is called "anti-TgammaA". This monoclonal antibody belongs to the IgG 2 sub-class. This monoclonal antibody can be labelled with a suitable marker such as radio label or a fluorescent marker for identif;ying and WO 89/02899 PCT/US88/03123 -4defining the sub-population of human T lymphocytes in circulating blood drawn from healthy individuals, ill individuals or individuals having been subjected to an organ graft or a bone marrow transplant. The antioody is also useful for characterizing th state of differentiation of human T lymphocytes in circulating blood.

Further, the antibody may be applied therapeutically by in-vitro, ex-vivo or in-vivo eventual manipulations of human T lymphocytes bearing the TigammaA epitope.

Disclosure of Invention A hybridoma cell line which produces a monoclonal antibody which specifically binds a unique epitope of the human T cell surfabe receptor of T lymphocytes in peripheral blood encoded by a T cell gamma rearranging gene. The epitope is located in a glycoprotein structure on the surface of human T lymphocytes in circulating bl6od having a molecular mass of approximately 85,000 daltons determined under non-reducing conditions and of 41,000-44,000 daltons determined under reducing conditions. The monoclonal antibody of the invention is called "anti-TigammaA" and belongs to the IgG 2 subclass. This monoclonal antibody enables characterization of human T lymphocytes in circulating blood which express the TigammaA epitope or antigenic determinant.

Best Mode for Carrying Out the Invention The monoclonal antibody anti-TigammaA binds to an antigenic determinant of the human T lymphocyte receptor and specifically to the Ti complex of the T 3 Ti structure of the human T cell surface receptor. The Ti molecular structure bears the gamma chain and the anti- TigammaA monoclonal antibody defines the gamma chain so that the invention enables detection, quantitation and thorough characterization of the Ti complex of the "second family" of the T cell surface receptor. This epitope appears on a sub-population of T lymphocytes in human blood and in human lymphoid organs, such as the thymus, the spleen, the ganglions and the bone marrow.

The advantages capable of being derived from the diagnostic and therapeutic applications of this monoclonal WO 89/02899 PCT/US88/03123 antibody will be amplified herein.

The anti-TigammaA monoclonal antibody of this invention was developed as hereinafter described.

A suspension of cells of a human lymphocyte clone, of the CD3 WT31- phenotype, "Nowill et al., Natural clones derived from fetal (25wk) blood, J. Exp. Med.

63:1601 1986", is prepared, which cells do not express on their surface the T a/f receptor. These cells express a functional activity called "natural cytotoxic activity". The suspension is injected into a Biozzi mouse. Immunization is carried out by two first intraperitoneal injections of 4 x 106 cells in a Freund adjuvant, followed by an intravenous injection of 4 x 106 cells without adjuvant. Three days later, the animal is killed, the spleen is drawn and the spleen cells are harvested.

Hybridization is then carried out by fusing the harvested spleen cells and a murine NS-1 myeloma cell line. Such a cell fusing has been carried out according to a known process disclosed in "Moingeon P et al., 1986, Nature, Vol. 323, p. 638". The hybrid cells obtained after fusing are cultivated in a HAT medium containing hypoxanthine, aminopterin, thymidine, 10% fetal bovine serum and 10% horse serum.

The hybridomas are Then selected. The screening is carried out by testing the ability of hybridoma supernatants to block cytotoxic reactions exerted by the clone immunizing against a leukemia cell line maintained in the culture.

The hybridoma producing the monoclonal antibodies are cloned by techniques of limiting dilution. Finally, immune ascites are produced, wherein the antibodies can be taken. The so-made anti-TigammaA antibodies belong to the IgG 2 subclass and can fix complement 35 protein.

The reactivity of the monoclonal antibody and the extent of its action have been determined with a subpopulation of human lymphocytes by means of two color immunofluorescence analysis and reading with i WO 89/02899 PCT/US88/03123 -6cytofluorometric methods. The cells of the subpopulation to be tested have been isolated from the mononuclear portion of the peripheral blood taken from healthy adult donors.

This portion has been purified by a centrifugation technique on Ficoll gradient. Samples of 3.0 x 105 cells are analysed on a cytofluorometer, such as an "EPICS C" flow cytometer manufactured by Coulter Corporation of Hialeah, Florida, U.S.A. The control reagents are FITC-IgG and PE-IgG.

The non-adherent lymphocytes are treated during minutes with the monoclonal antibody of the invention, anti-TigammaA, provided with a fluorescent green markers, namely fluorescein, in presence of another known antibody which is also provided with a fluorescent red marker, which is the phycoerythrin The other known antibodies are the following: anti-CD2, -CD3, -CD4, -CD5, -CD8, NKH1, and those antibodies determining the molecules encoded by genes called the class II genes of the major complex of histocompatibility. These known antibodies are provided by the company Coultronics, France of Margency, France.

The results obtained from the antibody according to the invention allow to characterize human T lymphocytes expressing the TigammaA antigenic determinant.

The great majority of TigammaA+ lymphocytes express CD2 and CD3 glycoproteins. A majority of TigammaA+ lymphocytes express CD5 proteins. It is to be observed here that, where these CD5 proteins are present, the density of the CD5 antigen per cell is lower in the TigammaA+ cell subset than in the population of conventional lymphocytes expressing the T a/P receptor. The expression of CD8 in the TigammaA+ subpopulation varies widely from an individual to the other. In most of the individuals, only a minority of TigammaA+ lymphocytes express the CDS protein with a weak antigenic density.

At rest, the TigammaA+ lymphocytes do not express the CD4 proteins, NKHI and molecules encoded by genes j i i

-I

WO 89/02899 PCT/US88/03123 -7of the class II of the major complex of histocompatibility.

Other comparative analyses of the reactivity of the anti-TigammaA monoclonal antibody, carried out by means of immunofluorescence techniques, have allowed to detect the cells expressing the CD3 proteins on the one hand, and the calls expressing at T receptor on the other hand, by using available T 3 specific monoclonal antibody and the BMA 031 monoclonal antibody (supplied by Behring These particular antibodies have been chosen, because they prevent optimal signals in indirect immunofluorescence analysis.

The results show that the TigammaA+ sub-population represents a majority of circulating lymphocytes which express CD3 proteins but which do not express on their surface the conventional T receptor.

On the other hand, certain functional effects induced by the interaction between the anti-TigammaA monoclonal antibody and the lymphocytes which express the corresponding antigenic determinant, have been studied.

In the presence of exogeneous Interleukin 2 with amounts of about 50 U/ml NIH, the interaction of the monoclonal antibody with the antigenic determinant leads to the activation and proliferation of the noncultivated circulating lymphocytes.

When the TigammaA+ lymphocytes are pre-activated with a mitogen such as e.g. the phytohemagglutinin with a dose of about 2 mg/ml, the cultivated and stimulated with the anti-TigammaA monoclonal antibody coupled with sepharose beads activated by the cyanogen bromide, a proliferation of the T lymphocytes is clearly shown 48 hours after the stimulation. These T lymphocytes further can secrete their own Interleukin 2.

The cytotoxic activity of the lymphocytes has been observed more precisely by means of other experiments.

The cytotoxicity is measured during an experiment of three hours after marking of target cells by 51 Cr.

The TigammaA+ cells determined by the monoclonal WO 89/02899 pcT/Uss88/3123 -8-

I

antibody of the invention, appear, when at rest, as a very homogeneous population of granular lymphocytes.

However, they mediate a little or no spontaneous cytotoxic activity against the conventional targets of the system called "natural cytotoxic system", as for example the K562 line which derives from a pleural effusion of chronic myeloid leukemia.

Consequently, this sub-population does not significantly contribute to the natural cytotoxic activity of the peripheral blood, and this is compatible with the absence of expression on the surface of the NKH1 marker, as explained previously.

On the contrary, when the TigammaA+ lymphocytes are activated by means of mitogens and by exogeneous Interleukin 2, they become very cytotoxic against numerous tumor cells, in particular those cells of the K562 line. This cytotoxicity can be induced within a very short time period from 3 to 5 days. The so activated TigammaA+ lymphocytes may then participate to the phenomenon called "lymphokine activated killer function" (which corresponds to the British "LAK phenomenon").

Furthermore, following the interaction between activated TigammaA+ lymphocytes and the anti-TigammaA monoclonal antibody, an important modification of the cytotoxic function is observed.

The type of modification depends on the experimental conditions used.

If one uses relatively resistant target cells expressing the Fc receptor of immunoglobulins, as e.g.

the U 937 human histiocytic line, the action of the antibody results in a substantial increase of the cell cytotoxicity.

Such results are explained by 'the fact that the anti-TigammaA antibody can bind the Fc fragment expressed on certain target cells. More precisely, in such a binding., the antibody establishes a bridge between effector and target cells and modifies the TigammaA antigenic determinant. Consequently, the lytic i- i' 1

SB

SWO 89/02899 PCT/US88/03123 -9process is triggered.

On the contrary, if one chooses target cells deprived of receptor for the Fc fragment of immunoglobulins, as e.g. the leukemia cell line called "jurkat", the action of the antibody results in a virtual abrogation of the cytotoxic function.

As a matter of fact, in the case where the target cells do not express the Fc receptor, the reaction of bridge binding disclosed above for the U 937 human histiocytic cell line, does not occur. Here, the antibody interacts only with its specific antigenic determinant through the Fab fragment. This interaction induces a modulation of the CD3-TigammaA molecular complex, the CD3 and TigammaA molecules being noncovalently linked to the cell surface. This phenomenon results in an important decrease of the cell lysis.

It is to be observed here that all the results previously described directly lead to the hypothesis that TigammaA represents a portion of a functional receptor involved in mechanisms of cell cytotoxicity which is not restricted by the genes of the major complex of histocompatibility.

Some immunoprecipitation experiments performed with the anti-TigammaA antibody have shown that the structure of the receptor characterized by the antibody, is in fact a molecular complex associated to CD3 proteins, this complex being bound by disulphide links.

These immunoprecipitation experiments further allow to identify the molecular weight of the antigenic determinant defined by the anti-TigammaA antibody.

The experiments are conducted with a fetal human cell line known under the name F6C7. These F6C7 cells are marked on their surfaces by 1251 by using a standard lactoperoxidase method, which is disclosed in "Moingeon 1986, Nature, Vol. 3 2 3 p. 638".

Several steps of pre-clearing are carried out with a suspension of Staphylococcus A, and also with other antibodies. Then, reactions of precipitation take .WO 89/02899 PCT/US88/03123 place during about 4-6 hours at a temperature of 4 0

C

with anti-TigammaA monoclonal antibodies, coupled to beads of sepharose-protein A. The SDS-PAGE electrophoresis is carried out on a 10% polyacrylamide gel under non-reducing conditions on the one hand, and under reducing conditions after addition of 5% of 2-ME, on the othe hand.

The results of the electrophoretic analysis are the following.

The TigammaA molecule migrates and produces an homogeneous band at 85,000 daitons approximately under non-reducing conditions. With reducing conditions, the molecule appears as a major band at about 44,000 daltons and a minor band at about 41,000 daltons. This immunoprecipitation pattern is very analogous from one cell to the other. Minimal differences of electrophoretic mobility, from one cell to another, probably correspond to differences of glycosylation.

These experiments show that the two bands of 44,000 and 41,000 daltons immunoprecipitated by the anti-TigammaA antibody correspond to proteins totally and partially glycosylated an which are encoded by the T cell gamma rearranging gene.

The precipitated gene encoding the TigammaA protein on the cell surfEce is more precisely characterized by a series of experiments called "Southern and Northern blot" using several appropriate molecular probes such as probes of the C gamma, J gamma and V gamma type. The results of these experiments have led to the following conclusions.

The specific rearrangement resulting in the transcription of the RNA messenger which provides the TigammaA protein, involves a unique junction between the V gamma 9 gene and the J gamma P gene. This rearrangement is further transcribed with the C gamma 1 constant region, because the protein expressed at the cell surface is a dimer bound by disulphide bridges.

Thus, the anti-TigammaA monoclonal antibody recognizes a glycoprotein structure, encoded by a recombined gene T l 1 /03l28---- 8 8./0312 IPE J 030CT1989 associating a V gamma 9 segment to a J gamma P segment.

All the previously described experiments and results characterize the anti-TigammaA monoclonal antibody and consequently, the receptor structure of the T lymphocyte cells which is associated to CD3 proteins and involved in the cell functions, in particular the cytotoxic functions.

The hybridoma cell line pD ocsiing the anti- TigammaA antibody is deposited in the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Maryland, 20852, as of September 1987, under ATCC No. HB 9528.

The monoclonal antibody according to the invention can be used to identify the human T lymphocyte cells bearing a receptor encoded by the gamma genes. It can also be used to characterize the state of differentiation of these T cells.

Moreover, this monoclonal antibody can be applied therapeutically against auto-immune diseases, carcinoma and viral infections. The monoclonal antibody used exvivo could allow the depletion of corresponding lymphocyte populations before organ or bone marrow transplants. It is also contemplated to use the antibody after transplantation procedures in-vivo for mediating against either the graft rejection, or the graft versus host disease, in the event it is determined that the cells expressing TigammaA play a part in these biological phenomena.

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Claims (13)

1. i monoclonal antibody of the anti-TigammaA type, characterized in that the said antibody specifi- cally binds to a receptor of the T lymphocytes of the "second family" encoded by genes called "gamma" in the V or variable region of said gamma gene.

2. A monoclonal antibody according to claim 1, characterized in that it belongs to the sub-class of IgG2.

3. A ionoclonal antibody according to claims 1 or 2, characterized in that it specifically binds to a glycoprotein structure on the surface of human T lymphocytes having a molecular weight of about 85,000 daltons such as determined by SDS- -3E electrophoresis under non-reducing conditions.

4. A monoclonal antibody according to claims 1 or 2, characterized in that it specifically binds to a glycoprotein structure on the surface of human T lymphocytes having a molecular weight of about 41,000 to 44,000 daltons as determined by SDS-PAGE elec- trophoresis under reducing conditions.

A monoclonal antibody according to claims 1 or 2, characterized in that it specifically binds to a glycoprotein structure, encoded by a recombined gene associating a V gamma 9 segment to a J gamma P segment.

6. A monoclonal antibody of the anti-TigammaA type, characterized in that it is produced by a line of hybidoma cells as deposited at the ATCC under the No. HB 9528.

7. A monoclonal antibody according to any one of claims 1 to 6 characterized in that it has a detectable label conjugated thereto.

8. A monoclonal antibody according to claim 7 characterized in that said label comprises a raddioac- tive or fluorescent marks'

9. A hybridoma cell line produced by hybridoma technique, characterized in that it is capable of producing a monoclonal antibody of the anti-TigammaA type which specifically binds to a receptor of the T lymphocytes of the second family encoded by genes S ,"SisrJTE Si1ET 'VEA/US ii; PCT,~~ 88/0312 -r; PeT/US 8 0.'/01 -13- iPE!.I"2 03 OCTIg8 called gamma in the V or variable region of said gamma gene. A hybridoma cell line produced by hybridoma technique, characterized in that it has the identifying characteristics of the sample on deposit with the Amer- ican Type Culture Collection No.

HB 9528 producing a monoclonal antibody of the anti-TigammaA type.

11. The use of a monoclonal antibody such as defined according to any one of claims 1 to 6, for im- munoprecipitating molecules having a T receptor struc- ture associated to CD3 proteins.

12. The use of a monoclonal antibody such as defined according to any one of claims 1 to 6, for im- munoprecipitating membrane molecules having molecular weights of 41,000 to 44,000 daltons and 85,000 daltons as determined under reducing and non-reducing condi- tions, respectively. gs^F NC 4 !I1V~ r I 4 INTERNATIONAL SEARCH REPORT International Application No. PCT/US8 8 /03123 I. CLASSIFICATION OF SUBJECT MATTER (il several classification symbols apply, Indicate all) 6 According to International Patent Classification (IPC) or to both National Classification and IPC IPC(4): C07K 15/04; C12N 5/00 U.S. CL.: 530/387; 435/ 240.27 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols U.S. 530/387,395,413 935/100,104,108 435/240.27, 68, 172.2 436 539 548 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched a Databases: Automated Patent System (File USPAT, 1975-12/88), Chemical Abstracts Online (File CA 1967-12/88); File Biosis

1969-12/88). See attachment for Search Terms. III. DOCUMENTS CONSIDERED TO BE RELEVANT Category Cilalion of Document, it with indication, where aprpropriate, of the relevant passages 12 Relevant to Claim No,

13 X Nature, Volume 325, Issued 19 February 1-4,12,13 Y 1987, P. Moingeon et al, "A gamma-chain 7-1( complex forms a functional receptor on cloned human lymphocytes with natural killer-like activity," see abstract, page 723. X,P Journal of Experimental Medicine, 1-6,U0-13 Y Volume 166, Issued October 1987, 7-9 S. Jitsukawa et al, "A Novel Subset of Human Lymphocytes with a T Cell Receptor-Gamma Complex," see pages 1192 and 1193. X,P Journal of Experimental Medicine, 1-6,11 Y Volume 167, Issued February 1988, 7-10,12,13 F. Triebel et al, "A Unique V-J-C- Rearranged Gene Encodes a Gamma Protein Expressed On the Majority of CD3+ T Cell Receptor-g/- Circulating Lymphocytes," see pages 698 and 699. Special categories of cited documentsa to later document published after the International filing date "A document deing the general state of the art which Is not 0or priority date and not In conflict with the appllatlon but A" document dening the general sat the art whiccited to understand the principle or theory underlying the considered to be at particular relevance Invention earlier document but published on or after the international (X document ol particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throv doubts on priority clalm(s) or Involve an inventive step which is c'ted to establish the publication date of another document of particular relevance; the claimed Invention citation o, otier special reason (as .oecified) cannot be considered to Involve an inventive step when the document referring to an oral disclosure, use, exhibition or document Is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the International filing date but in the art. later than the priority date claimed document member of the same patent family IV, CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this international Search Report 29 December 1988 09FEB 1989 International Searching Authority ISlgn olfA orl Oficer SA/US KA CHENEY Form PCT/SAI2IO (ucond het) (Rv.11.87) PCT/US88/03 123 Attachment to PCT/ISA/210 Part II. Fields Searched Search Terms T-cell receptors, T-cell surzc~ce receptors, T-cell antigen receptors, gamma, CD3, T3, monoclonal antibodies pp r international Application No. T/US FURTHER INFORMATION CONTINUED ROM THE SECOND SHEET03123 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET Proceedings of the National Academy of Sciences, Volume 84, Issued June 1987, loannides, C.G. et al, "Identification of a Second T-cell Antigen Receptor in Human and Mouse by an Anti-Peptide Gamma-chain-specific Monoclonal antibody," see pages 4244, 4247 and 4248. 1,10,12 7-9 OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE I This internalional search report has not been established in respect of certain claims under Article 17(2) lor the following reasons; 1,1 Claim numbers ,because they relate to subject matter not required to be searched by this Authority, namely: Claim numbers because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out specifically: Claim rumbers because they are dependent claims not drafted In accordance with the second and third sentences of PCT Rule 6.4(a). VI,[ OBSERVATIONS WIERE UNITY OF INVENTION IS LACKING This International Searching Authority found multiple Inventions in this International application as follows; As all required additional search fees were timely paid by the applicant, this International search report covers all searchable claims of the international application. As only some of the required additional search fees were timel, paid by the applicant, this International search report covers only those claims of the International application for which fees were paid, specifically claims: No equlred additional search fees were timely paid by the applicant. Consequently, this international search report Is restricted to the Invention first mentioned In the claims; II Is covered by claim numberst 4.J As all searchableclatms could be searched without effort justifying an additional fee, the International Searching Authority did not Invite payment of any additional fee. Remark on Protest t The additional search lees were accompanied by applicant's protest. Q No protest accompanied the payment of additional search fees, FormI PIT/ SA210 (upplrmal sheet (Fev. 11-87)