AU623345B2 - Human anti-rh(d) monoclonal antibodies - Google Patents

Description

AU-AI-25212/88 PCT WORLD INTELLECTUAL PROPERTY ORGANIZATION INTERNATIONAL APPLICATION 6L 3D 3 T4P AT T COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 89/ 02442 C07K 15/00, C12N 5/00, 15/00 C12P 21/00, A61K 39/395 Al G01N 33/80 (C12P 21:00 (43) International Publication Date: 23 March 1989 (23.03.89) C12R 1:91) (21) International Application Number: PCT/GB88/00755 (74) Agents: HOLMES, Michael, John et al.; Frank B.

Dehn Co., Imperial House, 15-19 Kingsway, Lon- (22) International Filing Date: 16 September 1988 (16.09.88) don WC2B 6UZ (GB).

(31) Priority Application Number: 8722018 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (32) Priority Date: 18 September 1987 (18.09.87) pean patent), DK, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (Eu- (33) Priority Country: GB ropean patent), NL (European patent), SE (European patent), US, (71) Applicant (for all designated States except US): CEN- TRAL BLOOD LABORATORIF AUTHORITY Published [GB/GB]; Dagger Lane, Elstree, Borehamwood, Hert- With international search report.

fordshire WD6 3BX Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt (72) Inventors; and of amendments.

Inventors/Applicants (for US only) DE BURGH 89 BRADLEY, Benjamin, Arthur [GB/GB]; Follybrook 2 M 1 Cottage, 12 Bury Hill, Winterbourne Down BS17 lAD DOYLE, Alan [GB/GB]; 12 Falcons Way,

AUSTRALIAN

Salisbury SP2 8NR KUMPEL, Belinda, Mary [GB/GB]; Woodside, Wrington Road, Congresbury, 17 APR 989 Bristol BS19 5AR (GB).

PATENY OFFIC( (54) Title: HUMAN ANTI-Rh(D) MONOCLONAL ANTIBODIES (57) Abstract The present invention provides human monoclonal antibodies having the following essential characteristics: exhibiting activity against Rh(D) antigen, but not C, c, E ore antigens of the Rh blood group system: being IgGI proteins; having kappa light chains; being Glm or Glm 17) allotype; exhibiting activity against Du cells by an indirect antiglobulin test: exhibiting activity against DIV, D v and DTar variant antigens; and being inactive against DVI or DB variant antigens, wnich may be employed for Rh-typing o. red blood cells and passive immunization to prevent haemolytic disease of the newborn, Cell lines capable of producing suoh monoclonal antibodies were deposited at the European Collection of Animal Cell Cultures, Porton Down, U.K. On 16th September 1987 under accession numbers ECACC 86091605 and ECACC 86091604, i 14 WO 89/02442 PCT/GB88/00755 1 Human Anti-Rh(D) Monoclonal Antibodies The present invention relates to human monoclonal antibodies to the Rh(D) antigen of human red blood cells. In particular, it relates to such antibodies of the IgGi sub-class which may be used to detect not only the normal Rh(D) antigen on either D-positive or "weak D" or Du cells, but also important variants of the Rh(D) antigen.

Of the antigens of the so-called Rh blood group system, the Rh(D) antigen is responsible for some of the most severe reactions following transfusion to a patient with corresponding antibody.

Since an Rh(D-) individual with anti-Rh(D) who receives Rh(D+) blood is liable to suffer substantial red blood cell '(RBC) destruction due to the Rh(D) phenotype incompatibility, blood of donors and blood transfusion recipients is routinely classified as Rh(D+) or Rh(D-) by agglutination tests with anti-Rh(D) antibody. The Rh phenotype of RBCs I is commonly further defined with reference to the Fisher-Race system, which is based on the assumption that the inheritance of the Rh antigens is determined by three pairs of allelic genes, C-c, D-d and E-e, acting at very closely linked loci. According to this theory, a person may inherit a set of three Rh genes from each of his parents C or c, (ii) D or d, (iii) E or e (no d antigen has as yet been identified, but the symbol is used to indicate the presence of a gene, allelic to the D gene, which does not produce D antigen). For example, an Rh(D+) person may inherit CDe from one parent and cde from the other. The frequencies of the commonest Rh gene combinations as determined with I i. C_ i i WO 89/02442 PCT/GB88/00755 2 reference to the Fisher-Race system for an English population, together with the 'short symbols' which are used, particularly in speech, are given in Table 1 below.

Table I Frequency of common Rh genes for an English Population Short symbol CDE nomenclature Frequency

R

1 CDe 40.8 r cde 38.9

R

2 cDE 14.1

R

0 cDe 2.6

R

lw CwDe 1.3 r" cdE 1.2 r' Cde 0.01 Rz CDE rarer Y CdE

=N

R (C)D(e) Despite expansion over the years, the Fisher- Race system has not been adequate to account for all the reactions that have been observed with the Rh system (MolLison, P.L. (1983) Blood Transfusion In Clinical Medicine, 7th edn., Blackwell Scientific, Oxford). Nevertheless, the World Health Organisation has recommended that in the interest cf simplicity and uniformity this nomenclature should be universally adopted and all Rh genotypes given hereinafter are defined on the basis of the conventional Fisher- Race system.

In addition to the need for anti-Rh(D) antibody for Rh-typing of RBCs, such 'antibody is also importantly required for passive immunisation of Rh(D-) mothers to prevent haemolytic disease of the newborn (HDN).

This condition arises in newborn Rh(D+) infants of Rh(D-) mothers previously sensitized to Rh(D) WO 89/02442 PCT/GB88/00755 3 antigen as a result of IgG anti-Rh(D) antibodies crossing the placenta during pregancy and causing foetal RBC destruction. Sensitization of the Rh(D-) mother to Rh(D) antigen may have occurred at the birth of an earlier Rh(D+) child due to some foetal RBCs entering the maternal circulation and being recognised by the maternal immune system. To reduce the incidence of HDN, it is routine practice in the United Kingdom and many other countries to give anti-Rh(D) antibodies to Rh(D-) mothers immediately after the birth of an Rh(D+) infant so that any Rh(D+) RBCs which have entered the maternal circulation are rapidly removed (Mollison, P.L. (1983) loc.

Scit.; Laros Jr., R.K. (1986), "Erythroblastosis Fe:alis" in "Blood Group Disorders In Pregnancy", Ch. 7, p. 103).

At the present time, anti-Rh(D) antibody for use in both Rh-typing of RBCs and passive immunisation of Rh(D-) mothers is largely obtained directly from female donors immunised during pregnancy or from immunised male volunteers. The success of the programme of post-partum prophylactic administration of human anti-Rh(D) immunoglobulin to Rh(D-) women has, however, resulted in a dramatic reduction in the number of naturally alloinmunised women (Urbaniak, "RhD haemolytic disease of the newborn: the changing scene", Br. Med. J. (1985) 291, Also, deliberate immunisation of individuals with Rh(D+) RBCs carries tihe risks common to receiving any transfusion of RBCs,e.g. risk of transmission of hepatitis viruses and HIV. Hence, there is much interest in obtaining human monoclonal anti- Rh(D) antibodies for both diqgnostic and therapeutic purposes.

As stated above, in routine blood testing, blood types are divided into Rh(D+) and Rh(D-) on the basis of the apparent presence or absence i- I i pC~ WO 859/02442 PCT/GB88/00755 4 of Rh(D) antigen on the RBCs as indicated by agglutination tests with anti-Rh(D). However, a small number of persons with apparently Rh(D-) blood have RBCs that are not directly agglutinated by anti-Rh(D) during such routine testing, but that do react when the D-typing test is performed using selected anti-Rh(D) reagents by the indirect antiglobulin test. Cells thus identified are designated DU The frequency of the D u phenotype is about 0.2% overall, 0.6% among Caucasians, and about of all Rh(D-) gravid women. At least three different mechanisms may be responsible for the expression of the D u phenotype: hereditary absence of a portion of the complete Rh(D) antigen, gene interaction with suppression of D by C in the trans position, and a D gene producing a weak antigen.

In the early 1950s, reports first appeared of the presence of anti-Rh(D) in individuals of the Du phenotype following blood transfusion with Rh(D+) blood or pregnancy resulting in the birth of a Rh(D+) infant. It later became apparent that in some individuals whose blood is classified Rh(D+) parts of the Rh(D) antigen are missing from the RBCs. When exposed by transfusion or pregnancy to Rh(D+) RBCs carrying the complete Rh(D) antigen, persons carrying an incomplete Rh(D) antigen on their RBCs are capable of making alloanti-D against the Rh(D) antigen portion they lack. The blood of such individuals is called D variant when the RBCs react directly with routine anti-Rh(D) reagents or Du variant when the cells react only by the indirect antiglobulin technicue.

The observation that allo anti-Rh(D) can be produced in patients who have Rh(D+) RBCs has led to common usage of the term "D mosaic" to describe the Rh(D) antigen in its complete native form.

Routine anti-Rh(D) reagents generally cannot differentiate those RBCs that lack part of the D mosaic from those that have all the D components.

The D variant phenotypes have been categorised by Tippett and Sanger (Vox. Sang. (1962)7, 9-13); This system is based on the interaction of RBCs and serum from D- and Du variant individuals.

The seven categories (see Table II below) allow for expansion; subdivisions are already recognised in categories III, IV and V. Categories I and TI have been found to have so many similarities that they are now generally considered as a single sub-group.

Table II Tippett and Sanger categories for D- or D positive blood with anti-Rh(D) Category' Racial origin Usual haplotyt.

I White DCe

II

IIIa Black IIIb Usually Black Dce IIIc White IVa Mostly Black, some White IVb White Dce Va Black and White Vb White D Ce Vc Black and White VI Nearly all White DCe VII White DCe An alternative, but lesser used, classification by Wiener uses letters A,B,C,D instead of Roman Numerals. Although there is no dirt-t correlation between the two systems, it is often considered that D and D V 1 are interchangeable.

Although the frequency of D and Du variant individuals within the human population is relatively WO 89/02442 PCT/GB88/00755 6 low, the total number of individuals of these blood types who potentially have some risk of effective anti-Rh(D) formation as a result of exposure by blood transfusion or pregnancy to non-variant Rh(D+) cells is far from insignificant. Moreover, in addition to Rh(D-) women who give birth to Rh(D+) or Du infants, Du variant women who give birth to an Rh(D+) infant may also benefit from postpartum anti-Rh(D) treatment to reduce the risk of HDN (White, C.A. et al. (1983) Am. J. Obstet.

Gynecol. 145, 1069-1073). Anti-sera capable of distinguishing D and Du variant RBCs are not widely available. Hence, provision of anti-Rh(D) monoclonal antibodies with a range of binding specificities for D and Du variant RBCs is seen as useful in enabling the more ready identification and categorisation of individuals possessing such cells (especially D or Du variant pregnant females who are suitable candidates for prophylactic anti-Rh(D) treatment) as well as for proiding further structural information on the Rh(D) antigen complex.

Human monoclonal anti-Rh(D) antibody production has previously been achieved by:directly cloning Epstein Barr virus transformed B lymphocyte cell lines (hereinafter referred to as EBV-transformed LCL) derived from B lymphocytes of anti-Rh(D) positive donors (see GB-A 2127434; Crawford et al. (1983) Lancet 1, 386-388 and Paire et al (1986) Immunol. Lett. 13, 137-141), cloning hybridoma cell lines formed by fusing anti-Rh(D) producing, EBV-transformed LCL with mouse, mouse-human or human myeloma cell lines (see co-pending British application no. 8709748, Thompson et al. (1986) Immunol. 58, 157-160 and EP-A-0162918), or by fusion of a human LCL with immune B cells (Lowe et al (1986) Vox. Sang. 51, 212-216).

I I -I ;"39 7- By cloning EBV-transformed LCL from 3 anti- Rh(D) positive donors, we have been able to obtain, however, monoclonal anti-Rh(D) antibodies of the IgG class which have a particularly useful binding specificity spectrum not shown for any previously disclosed anti-Rh(D) monoclonal antibody reagent.

According to the present invention, we provide human monoclonal antibodies having the following essential characteristics: exhibiting activity against Rh(D) antigen, but not C, c, E or e antigens of the Rh blood group system; being IgGl proteins; having kappa light chains; being Glm or Glm 17) allotype; exhibiting activity against Du cells by an indirect antiglobulin test; exhibiting activity against DIV, DV and DVI variant antigens; and being inactive against D V or D variant antigens, and antigen-binding fragments thereof.

Such monoclonal antibodies can be employed as routine anti-Rh(D) reagents to classify RBCs as D u or Rh(D-).

For this purpose, a monoclonal antibody of the present invention may be employed either alone or in combination with one or more further anti- Rh(D) antibodies, preferably monoclonal antibodies, having one or more additional binding specificities.

Thus, for example, a monoclonal antibody of the present invention may be advantageously blended with a further monoclonal antibody capable of binding the DVI variant to provide an anti-Rh(D) reagent of broader specificity capable of classifying DVI variant RBCs as D-positive.

In such an anti-Rh(D) reagent, an IgGI antibody of the present invention may, for example, be combined L_ I

I_

8 with an IgG1 monoclonal antibody of the type disclosed in our co-pending International application (Publication No. W089/02443) of even date herewith having the following binding characteristics: exhibiting activity against Rh(D) antigen, but not C, c, E or e antigens of the Rh blood group system; exhibiting activity against D DII D and

B

D variant antigens; and being substantially non-reactive with non-papain treated DIV cells in an IAG test.

Amongst such antibodies, particularly preferred for E* use in combination with an anti-Rh(D) monoclonal antibody of the present invention is the monoclonal 15 antibody designated B7 deposited at the European Collection of Animal Cell Cultures, Porton Down, U>K> under accession No. ECACC 87091603 on 16th September S 1987.

If monoclonal antibodies of the present invention 20 are used for Rh-typing in parallel with an anti- Rh(D) I reactive against the DVI variant, eg. an appropriate polyclonal anti-Rh(D) serum, those blood samples giving a positive result in an agglutination test with the latter, but negative results with a monoclonal antibody 25 of the invention can be predicted to be mainly or entirely of the DVI category (since this is virtually the only D variant antigen against which the new monoclonal antibodies are inactive). It has been established that amongst individuals classified as Rh(D+) or D u by a conventional agglutination test, but who are capable of making anti-Rh(D), a high percentage have the D variant antigen (Mollison, P.L. (1983) in "Blood Transfusion In Clinical Medicine", Ch. 8, p 339).

One use of the monoclonal antibodies of the invention is, indeed, in investigating the incidence of individuals of the DVI type in the population.

L 2q9.

9 A monoclonal antibody of the present invention may also be of particular value for use in an anti- Rh(D) typing reagent to supplement the specificity of an anti-Rh(D) with no or only weak anti-D u activity, i.e. insufficient activity against Du cells to be able to reliably distinguish such cells from D-negative cells in a conventional agglutination test. Indeed, under FDA regulations in the U.S.A.

governing commercial anti-Rh(D) typing reagents, it is obligatory for such a reagent to be able to distinguish Du RBCs from truly D-negative RBCs.

Especially preferred amongst combination anti-Rh(D) reagents of the present invention are such reagents satisfying the above condition wherein an IgG anti- Rh(D) of the invention is employed together with an IgM anti-Rh(D) with no or only weak Du activity, e.g.

an IgM monoclonal anti-Rh(D) selected from the monoclonal IgMs of the deposited hybridoma cell lines MAD-2 (ECACC 86041803) and FOM-1 (ECACC 87021301), which form inter alia, the subject matter of published European Patent Application 0251440.

Such a reagent will advantageously be further complemented with at least one further IgG monoclonal anti-Rh(D) antibody which individually exhibits activity with

D

u red cells by the indirect antiglobulin test, such that the blended reagent reacts by the same test with D u

D

I

D

V and D V I cells. When Rh typing is carried out with such a reagent, D-positive cells will firtly be directly agglutinated by the IgM anti-Rh(D). The remaining non-agglutinated cells (apparently D-negative) may then be subsequently divided into truly D-negative and Du cells by addition of conventional Coomb's reagent for an indirect antiglobulin test, whereupon Du cells binding IgG antibody will be agglutinated and thus distinguished.

The monoclonal anti-Rh(D) antibodies of the S invention can be made by conventional methods known /i rrr r WO 89/02442 PCT/GB88/00755 10 for the production of monoclonal antibodies and in particular by the culture of EBV-transformed human B-lymphocytes selected on the basis of secretion of anti-Rh(D) immunoglobulin having the characteristics set out above for the required antibodies. The culture supernatants so produced constitute a further feature of the present invention.

We have now investigated in detail 9 cloned EBV-transformed LCL which produce IgGI anti-Rh(D) monoclonal antibodies as defined above. All these cloned cell lines were obtained by starting with peripheval B lymphocytes from 2 chosen anti-Rh(D) donors and employing the procedure described in GB-A 2145113 or a substantially similar procedure to establish and clone EBV-transformed LCL producing a monoclonal antibody of the desired specificity (see Example In continuous culture using RPM1- 1640 medium supplemented with 10% mycoplasma free-fetal calf serum, 0.2 mg/ml arginine and antibiotics to prevent mIycoplasma growth, they have been found to be highly stable and to provide a culture supernatant having an anti-Rh(D) titre, as determined by an indirect antiglobulin (TAG) assay in low ionic strength saline versus RiR 1 (Ce/CDe) RBCs, in the range 2000-8000. Such a culture supernatant is suitable for use in Rh-typing without the need for concentration and indeed may be diluted for use. Four of the selected cell lines (Al, A2 and A3 derived from a donor A) have been shown WO 89/02442 1 WO 8902442P CT/C B88/00755 11 to miaitain a -stable anti-RhC(D) titre in continuous culture for over 2 years. Four further cell line, capable of providing a culture supernatant as above (81l, B2, B3 arnd Bll derived from a donor B) have been maintained in continuous c ,ulture for over 8 months without substantial decrease of anti-Rh CD) t,',tre. Antibody production characteristics of the above-mentioned specific clones in continuous culture are summarised in Tables Ilia and IIIb below.

Table lia TAG titre RBCs in low ionic strength saline) Rir cells 64-512

R

1

R

1 cells 2000-8000 P, r cells 8-512.

Microtitre with bromelain-treated R R 2 el 33,000-13l,000 Anti-Rh CD) 10-98 (lU/nil) IgG 1.

I i I WO 89/02442 PCT/GB88/00755 12 Table IIb culture Al A2 A3 Bl B2 B3 Bi). B12 B13 Supernatant IAG reactivity of supernatant with 3% RBCs in 16w ionic strength saline.

(Grade: 0 to 6)

RBC

phenotype

R

1

R

1 6 6 6 6 6 6 6 6 6 R lir 6 6 6 6 6 4 5 6 6 RUr 6 6 6 6 6 6 6 6 6 It was further found that those monocional antibodies of the invention that were tested reacted by IAG with RBCs of phenotypes

R

2 TrG-, hrt, R R and R 2 R but are negative with r"Gr, r rG Os hr B rYwr and Rh33+.

Gm allotyping of the antibodies of clones Al, A2, A3,, I, 12, B3, B11, B12 and B13 1?as shown that they fall into two allotype groups, tUs:.nq the WOHOD. (1974) notation, the Tg 9 i anti-Rh(.W antibodies of clones Al, A2 and A3 were found be of the Glm 17) allotype, whereas the antibodies of the remaining above-mentioned clones derived from dtanor B were found to be of the allotype Glm(3).

The latter antibodies are of particular interest from the point of view of providing a monoclonal antibody preparation for use in post-pirtum immunisation of hD-) mothers. In general, the antibodies in the clm 17) allotype group were active against the V antigen, whereas those of Glm(3) allotype were not.

IgG1 anti-RhO antibodies are generally poor promoters of phagocytosis of Rh(D+) RBCs by monocytes C SV4 89/02442 PCT/GB88/t0755 13 3 macrophages. However, we have found that IgGi anti-Rh(D) antibodies of the Glm(3) allotype, e.g.

the IgGi antibodies of clones Bl, B2, B3, BL1, B12 and B13, unlike IgGi anti-Rh(D) antibodies of the Glm(1,17) allotype, are highly effective in mediating lysis of sensitised RBCs by K lympho es in an antibody-directed cell cytoxicity (ADCC) assay. This is in keeping with the previously reported observation of Parinaud et al. that foetal haemolysis is more severe with maternal IgG1 antibodies of the Glm(4) allotype, i.e. Glm(3) allotype according to the W.H.O (1974) nomenclature (Am. J. Obstet.

Gynecol (1985) 1111-1115).

Thus, according to a further aspect of the present invention, we provide a monoclonal IgGi antibody of the present invention, preferably a monoclonal antibody of the present invention having the allotype Glm(3), for use in passive immunisation of an Rh(D-) or D- or D u variant mother after the birth of an Rh(D+) child to prevent sensitisation of the mother to Rh(D) antigen. A sterile solution of such an antibody for human injection may be formulated in any physiologically acceptable aqueous medium, for example isotonic phosphate buffered saline or serum. Alternatively, the antibody may be supplied in a freeze-dried formulation ready for reconstitution prior to use. To provide a highly efficient prophylactic preparation for use in the prevention of HDN, a monoclonal anti-Rh(D) of the present invention, especially suchi antibody of the Glm(3) allotype, may desirably be employed with one or more further anti-Rh(D) antibodies, for example one or more further anti-Rh(D) antibodies promoting phagocytosis of Rh(D+) RBCs in vivo, e.g. an anti-Rh(D) monoclonal antibody of the IgG3 sub-class, such as an 1gG3 monoclonal anti- Rh(D) of our co-pending International Application 14 of even date herewith (Publication No. W089/02600) the contents of which are incorporated herein by reference.

The aforementioned IgG3 monoclonal anti-Rh(D) antibodies are exemp ified by the monoclonal antibody of the deposited cell line ECACC 87091606. For routine use, ideally a prophylactic pharmaceutical composition of the present invention will include an anti-DVI antibody.

According to a still further aspect of the present invention, we provide a method of Rh-typing of RBCs wherein an aqueous solution of a monoclonal anti-Rh(D) immunoglobulin of the present invention is employed.

The monoclonal immunoglobulin is preferably contained in a culture supernatant which may be used directly or, F more usually, after dilution. Particularly preferred 15 for use in Rh-typing are culture supernatants containing I monoclonal anti- Rh(D) immunoglobulin according tu the present invention which will agglutinate at high Sdilution 1:1000 dilution) enzyme-treated RBCs carrying the Rh(D) antigen and in the IAG test will i 20 agglutinate DU RBCs at, for example, 1:10 dilution.

As hereinbefore indicated, it may be desirable to blend an IgGl antibody of the present invention with one or more further anti-Rh(D) monoclonal antibodies of S" different specificity, e.g. a further IgG1 antibody S. 25 having anti-D activity. Suitable diluents include physiological saline or phosphate buffered saline, advantageously containing bovine serum albumin and a surfactant or suspending agent such as Tween 80 or methyl cellulose.

The cell lines A3 and B2 were deposited on 16th September 1987 at the European Collection of Animal Cell Cultures, Porton Down, U.K. under accession numbers ECACC 87091605 and ECACC 87091604 respectively.

Further details of the preparation of the above-noted depc ted cell lines of the present 1 i I WO 89/02442 PCT/GB88/00755 15 invention and the identifying characteristics of the culture supernatants obtainable by continuous culture of these are provided in Example 1 of the following non-limiting examples.

WO 89/02442 PCT/GB88/0075 -16 EXAMPLE 1 Establishment and Cloning of Anti-Rh Producing EBV-transformed LCL Sources of B Lymphocytes Donor A: female, imnmunised during her first and only pregnancy (which resulted in delivery of a normal Rh(D+) infant), boosted with 0.5 ml packed Rh(D+) (R 2 r) RBCs 4 years after parturition, and a peripheral blood sample obtained 8 days after boosting when her serum anti-D level was 601U/ml.

Donor B- male, initially immtunised by transfusion in 1966, boosted 6 times since and last boosted 13 days before donating a "buffy coat" fraction (white cells) in 1985 when his serum anti-D level wias 3181U/ml.

Establishment of cell lines Peripheral blood mononuclear cells from donor B were separated on Tymphoprep (Nyegaard and Co), incubated in the preseo'ce of EBV (I ml culture supernatant from filtered mycoplasma free B95-8 cell line per, 107 cells) at 37 0 C for 1 hour and washed in phosphate-buf fered saline (PBS) .Aliquots were either plated at 0.5x10 6 cells/ml in 2 ml wells using lymphoblastoid cell culture medium (RPMI- 1640 medium containing 10% mycoplasma free foetal calf serum (FCS) ,0.2 mq/r.nl arginine, 100 IU/nil penicillin (Glaxo) 50 pg/mi streptomycin (Glaxol IU/nd polymixin (Glaxo) 25 pg/ml kanamycin (Gibco) 20 p1/nil f ungizone (Squibb) 25 pg/nil gentamycin sulphate (Sigma) and 20 }iq/ml trobicin (Upjohn)) supplemented with either 1% ppytohaemaq- WO 89/02442 PCT/GB88/00755 17 glutinin (PHA) or 0.5 pg/ml cyclosporin A (CsA); or enriched for surface anti-D positive lymphocytes by rosetting with bromelain-treated OR 1

R

2 (CDe/cDE) RBCs prior to plating out as above.

Four cell lines from donor B were set up: LCL and enriched LCL with either initial PHA or CsA supplementation.

Isolated peripheral blood mononuclear cells from donor A were similarly infected with EBV.

After washing with PBS, the cells were resuspended in lymphoblastoid celi culture medium supplemented with 1% PHA and dispensed into 2 ml wells above a feeder layer of mouse peritoneal macrophages.

All the cultures were subsequently incubated at 37*C in 5% CO 2 95% humidified air. Medium changes were performed every 3 to 4 days and, after 3 weeks culture, the cells were transferred to ml flasks. All the cell lines (except that giving rise to clone B3) were enriched by rosetting at 3-4 weekly intervals.

Cloning Cells were plated out at limiting dilution at 5 and 10 cells per well in flat bottomed 96well plates over a feeder layer of mouse peritoneal macrophages (Doyle et al. (1985) Human Immunology 13, 199-209). Cultures were fed once a week and after 3-4 weeks cloned cells positive for anti- D were grown up.

Derivation of clones Three clones producing anti-Rh(D) antibody of the IgGl sub-class (designated Al, A2 and A3) WO 89/02442 PCT/GB88/0075,5 18 were derived from B lymphocytes of donor A. The polyclonal cell line giving rise to these clones was initiated with PHA and sequentially enriched for anti-Rh(D) positive cell by rosetting 13 times before cloning.

Five further clones producing anti-Rh(D) antibody of the IgGl sub-class (designated B 1

B

2

B

3 Bll and B12) were derived from B lymphocytes of donor B via a polyclonal cell line initiated with PHA. Bl and B2 were derived from cell lines repeatedly (6x) selected by rosetting after establishing an LCL. Bll and B12 were derived from a cell line rosetted only twice and B3 was produced from an LCL maintained for 1 year without rosetting before cloning. [It should be noted that Bll was erroneously designated Cl in the priority application (GB-A 8722018) A further anti-Rh(D) IgGI producing clone 'B13) was derived via a donor B polyclonal cell line supplemented with CsA. This cell line was established from lymphorytes selected for anti- Rh(D) immediately after transformation by EBV and was subsequently re-rosetted four times.

The results of tissue type and karotype analysis of the above selected cell lines are set out in Table IV below.

WO 89/02442 PCT/GB88/00755 19 Table IV Cell Line Al A2 A3 Bl B2 B3 Bll B12 B13 Donor A B Sex Female Male Tissue Type HLA: A 2,wl9 1,3 B 44,35 8,35 DR 3,5 1,3 Karotype sex chromosomes xx xy ploidy most cells most cells diploid diploid Quantitation of anti-Rh(D) activity and IgG in culture supernatants Anti-Rh(D) activity in the supernatants was quantified against British national standards by Auto Analyser. The mean of at least two determinations was calculated. The quantitative estimation of IgG was performed by ELISA (modification of the method of Wakefield et al. in Clin. Chim. Acta.

(1982) 123, 303-310) with at least eight determinations for each supernatant. Coating antibody (affinity purified goat anti-human IgG (Sigma)) was used at 1/200 in 0.05M carbonate buffer pH 9.6. Supernatants and standard (purified human IgG (Sigma)) were diluted in RPMI 1640 4 10% FCS. Peroxidase-conjugated goat anti-human IgG (Sigma) was diluted 1/500 in PBS 0.05% Tween 20 and the substrate was TMB 5,5'-tetramethyl benzidine).

Not all the cell lines established under different experimental conditions showed stable antibody production. However, all the cell lines 20 which were subsequently cloned had maintained high titres (over 1/33,000 by microtitre) of anti-D for over 6 months. All clones from these cell lines were positive for anti-D and maintained their titres throughout the duration of continuous culture (Al, A2 and A3 over 2 years; Bl, B2, B3 and B11 over 8 months). The doubling time was 3-7 days. The cells grew well in suspension culture with no loss of antibody production.

Immunoglobulin class and subclass determination An immunodot assay (McDougal et al. (1983) J. Imm.

15 Meth. 63, 281-290) was used to determine the reaction of the monoclonal anti-Rh(D) antibodies absorbed to nitrocellulose with anti-IgG, anti-IgM, anti-kappa and anti-lambda antiserum (Serotec); positive reactions were detected with peroxidase-conjugated anti-sheep IgG (Serotec) followed by colour development with 4-chloro-l-naphthol. The IgG subclass was evaluated by agglutination of anti-D coated RBCs by monoclonal '."anti-subclass antibodies (Unipath).

The presence of single discrete bands for antibody 25 derived from the selected cell lines after SDS-polyacrylamide gel electrophoresis was evidence of monoclonality.

SDS-Page and Western Blotting.

Iscove's supernatants (serum free) were electrophoresed under reducing conditions on polyacrylamide gels (Laemmli, Nature (1970) 227, 680-685). The separated proteins were then electrophoretically transferred to nitrocellulose membranes (Burnette, Annals Biochem. (1981) 112, 195-203), which were probed with anti-IgG antiserum (Serotec) and detected as above.

WO 89/02442 PCT/GB88/00755 21 Protein A absorption 2 ml volumes of supernatants were run twice down a 25 mm (iml) column of Protein A Sepharose C1-4B (Sigma) and absorption of anti-Rh(D) assessed by titration.

Gm allotyping RBCs were coated with the monoclonal anti- D antibodies and agglutination assessed using panels of Gm allotyping reagents (Birmingham or Amsterdam).

The Gm allotypes of the monoclonal antibodies of clones Al, A2, A3, Bl, B2, B3, B1l,, Bl2 and B13 together with other characteristics are given in Table V below.

WO 89/02442 WO 8902442PCT/G B88/00755 22 Table V Identification of iinmurioglobulii class, subclass. and Gm allot e monoclonal Al A2 A3 B B2 B 3 B12 B13 antibody: Anti-IgG: anti-IgM: 44+ 4+ +4-4 anti-kcappa: anti-lambda: Anti-IgGl: anti-IgG2:, ant i-IgG3: anti-IgG4: Heavy chain Mr (kDa): Protein A absorption Gmn allotype: 6 0 0 0 55.5 54 54 54 55 44+

GI

m I,17) tKi- 1 (71 m(1, 17)

KMI-).

m (1,17) G 1 m W-) m (3) G1 m (3)

G!

m (3)

GI

Mn(3)

GI

m (3) KM-I Kin-i Xm-i Km-i xInI-i not determined WO 89/02442 PCT/GB88/00755 23 (ii) Serology Anti-D titration was performed by adding pi of 0.1% suspension of bromelain-treated OR 1

R

2 (CDe/cDE) erythrocytes to 50 pl of supernatant, neat and serially diluted two-fold, in V-well microplates.

After incubation at 37°C for 60 mins, and centrifugation at 600 rpm for 3 mins, they were read macroscopically by tilting the plate at 70° and allowing negative reactions to trail. The degree of agglutination was graded in conventional manner and the titres given as the highest dilution giving complete agglutination (see Table lia). Anti-D titres were also determined by the IAG test using rabbit anti-human IgG and 3% Rlr, R iR, R r or Ror cells in low ionic strength saline (LISS). The anti-D activity of the monoclonals against a panel of D variant red cells was also assessed using saline, albumin, papain and IAG (in LISS) tests. In a separate series of tests, the reactivity of the monoclonal anti-Ds of Al, A2, A3, BI, B3 and Bll with Du red cells (15 sets of R 1 Ur cells and 10 sets of R 2 Ur cells, each set being taken from a different individual) was assessed by IAG (in LISS) using untreated supernatants.

Using the same procedure, culture supernatant of Bl was further compared with culture supernatant of B2 against 1 set of Rur cells and 3 sets of R r cells. The supernatants were tested on a Technicon Autogrouper 16C at dilutions ranging between to 1:10,000.

Tests against cells of "normal" RhD positive or RhD negative phenotypes by albumin, papain and indirect antiglobulin techniaues revealed that all the IgGl monoclonal antibodies showed specificity for the D antigen (see Table VI below). None of WO 8902442 PCTGB8800755 WI 089102442 PC/GB88/00755 fk I

I'

24 the monoclonal anti-Ds were reactive by saline.

When tested by IAG against "partial" D positive cells, the antibodies agglutinated DIV, DV and D red cells, but not D I or D B (see Table VII below). As shown in Table VIII, the supernatants of Al, A2, A3, Bl, B3 and Bl were all found to show stronger reactivity by IAG tests with 25 Du cells weak D) than polyclonal antiserum (the routine reagent employed by the South Western Regional Transfusion Service in the Using the same IAG test procedure, culture supernatant of Bl was found to exhibit similar or identical reactivity to culture supernatant of B2 against 4 further sets of Du cells (see Table IX). All the IgGl monoclonal antibodies reacted more strongly with l 1 r (CDe/cde) or RIR 1 (CDe/CDe) cells than with either D or D cells, though there were some differences in titres between the supernatants. When tested separately on a Technicon Autogrouper 160, supernatants of A2, BI and B11 could be used diluted to 1:1,000 for use as a routine anti-D and at 1:10 dilution to distinguish D u from D negative cells.

Table VT Serology of monoclonal anti-Rh(D) antibodies RBC RR 1

R

2

R

2 Ro r"r rr phenotype Saline 370 0Q 0 0 0 0 0 Albumin 37 0 C: 4-5 4-5 4-5 0 0 0 Papain 37 0 C; 5 5 5 0 0 0 IAG: 5 5 5 0 0 0 S(Grade 0 to (Grade. 0 to

I-

25 Table VII Reaction of monoclonal anti-Rh(D) antibodies with "partial" D positive RBCs by IAG Monoclonal antibody: Al A2 A3 BI B2 B3 B1] Di: 5 5 5 5 4 3 D 5 5 5 5 5 5 DVII: 5 5 5 5 5 5 D VI 0 0 0 0 0 0 0 D 0 0 0 0 0 0 0 (Grade: to 4 Table VIII Reactivity of monoclonal and polyclonal anti-Ds with DU RI3Cs byAG RU and 10 R 2 r) monoclonal Polyantibodyt Al A2 A3 Bi B3 311 clonal anti-Rh (D) Serum Number with Grade 6: 16 15 11 6 1 0 Grade 5: 8 9 12 16 7 17 4 Grade 4: 1 3 2 3 9 3 12 Grade3: 0 0 0 0 4 0 7 Grade 2: 0 0 0 0 4 0 3 rade It 0 0 0 0 0 0 1 r riii,,~ m~r: -f WO 89/02442 PCT1GB88/00755 25 Table IX Monoclonal Bi B2 antibody (titres by IAG) RBCs u RUr 512 512 1 R r 32 16 0 RU r 512 512 0 RQ r 52 512

Q

(iii) Lymphocyte ADCC assay Equal volumes (50 p1) of target cells (chromium-51 labelled R RIEBC suspension), effector cells (K lymphocytes) and anti-D culture supernatant were incubated overnight at 371C in microplates after mild centifugation, and the chromiur-51 release measured (Urbaniak (1979) Br. J. Haeiatol. 42, 303-314). The effetor:target cell ratio was 15:1.

The culture supernatants of clones Bi, B2, B3, B11i, B12 and B13 containig a monoclonal anti- Rh(D) of the 1in(3) allotype, unlike the other culture supernatants with antibody of the Glm(1,17' allotype, were found to be highly active in mediating lysis of sensitised RBCs in the presence of K cells (see Table X below).

L i WO 89/02442 PCT/GB88/00755 27 Table X Monoclonal Anti- Rh(D) antibody: Al A2 A3 B1 32 B3 Bll B12 B13 serum specific lysis: Effector KF -2 -1 -2 91 99 83 Effector KL 13 78 Effector BW 5 4 3 56 73 70 92 Effector BK 4 15 91 81 71 75 not determined (iv) U937 Monocyte rosetting and phagocytosis assay 100 l packed OR 1

R

2 RBCs were sensitised with' 500ul anLi-Rh(D) (previously adjusted to 1 ug/ml) at 370 for 60 minutes, washed and resuspended at 1 x 108 cells/ml in RPMI. U937.cells were taken in the log phase of growth and cultured for two days either in the presence or absence of interferon-) (Amersham) at 50 U/ml. 45 x 106 RBCs were then added to a pellet of 1.5 x 106 U937 cells and mixed, giving a ratio of 30:1. For the rosetting assay, the cells were incubated at room temperature for minutes, spun at 600 rpm for 3 minutes and examined in a haemocytometer after a further 5-20 minute'.

Phagocytosis was assessed immediately after incubating the cells at 370 for 3 hours. Results were expressed as the percentage of monocytes with one or more adherent or phagocytosed RBCs.

A comparision. of the results obtained wth culture supernatants of clones Al, A2, A3, Bl, B3 and B11 and a conventional polyclonal anti-Rh(D) serum is given in Figures 1 and 2.

I' WO 89/02442 PCT/GB88/00755 28 Macrophage binding assay RBCs (R 2 r) (1 volume) were sensitised with monoclonal anti-Rh(D) (2 volumes of untreated culture supernatant) and incubated with monocyte-derived cultured macrophages. The macrophages were stimulated with 500 U/ml of recombinant immune interferon (Biogen, Geneva) during the 48 hours prior to their use in the assay. The binding of RBCs to macrophages was assesed microscopically and expressed as the macrophage binding index number of red cells attached to or ingested by 100 macrophages).

Table XI below shows the results obtained with culture supernatants of clones AFl A2, A3, Bl, B3 and Bll. The ability of these supernatants to bring about RBC-macrophage interaction was very poor compared to that of the polyclonal anti-Rh(D) serum (anti-Rh(D) 43IU/ml) which served as a positive control.

Table XI Source of polyclonal anti-Rh(D) Al A2 A3 B1 B3 B11 anti- Rh(D) serum.

Macrophage 20 28 24 5 20 17 416- 500 binding index EXAMPLE 2 Solution for Rh(D)-Phenotyping of RBCs In general, it is preferred for the above purpose to use a blend of an anti-Rh(D) mono .nal antibody according to the invention B11; I I 14.

29 with a further IgG monoclonal antibody having anti-Dv1 activity, for example the monoclonal antibody of the cell line B7 (ECA'C 87091603) of our copending International patent application (Publication No.

W089/02443) of even date herewith, the contents of which are incorporated by reference into the present specification.

Solution for Manual Use The final blend is 1:1:1 Bll:B7: diluent.

Diluent 100 ml 30% Bovine Serum Alubumin 2.42g KH 2

PO

4 15 2.77g Na2HPO4.2H20 :4.50g NaC1 0.2 ml Tween 1.00g NaN 3 To 1.0 litre with distilled water: pH 20 6.8 This blend can be used in all manual tests for D and D u typing, e.g. microtitre, microplate, IAG.

25 A blend of 1:1:2 Bll:B7, diluent may also be used.

Blend for Machine Use This blend may be used, for example, in a Technicon autogrouper 16C Pre-blend reagents B11:B7 1:1 For D-Phenotyping (D-positive v. D-negative) the solution comprises 1:1000 blend: diluent, and for Du determinations (D u v. D-negative) the solution S comprises 1:5 blend:diluent.

I i I I I I 30 Diluent 2% Bovine Serum Albumin in 1.3% physiological saline containing 13.5% methylcellulose.

The monoclonal antibody B7 may be similarly blended with other antibodies of the present invention, e.g. the monoclonal antibodies of the deposited cell lines A3 (ECACC 87091605) and B2 (ECACC 87091604) to provide an anti-Rh(D) reagent with broad specificity for the various D-variant antigens, including D and exhibiting activity against Du cells by an IAG test.

z ~1 ~f xi J'4

Claims (13)

1. A human monoclonal antibody having the following essential characteristics: exhibiting activity against Rh(D) antigen, but not C, c, E or e antigens of the Rh blood group system; being IgGl proteins; having kappa light chains; being Glm or Glm 17) allotype; exhibiting activity against Du cells by an indirect antiglobulin test; exhibiting activity against DI, D and DI variant antigens; and being inactive against D or D variant antigens 15 and antigen-binding fragments thereof. S2. A monoclonal antibody as claimed in claim 1 selected from the monoclonal antibodies of the cell lines ECACC 87091605 and ECACC 87091604.

3. An anti-Rh(D) reagent wherein a monoclonal antibody as claimed in claim 1 or claim 2 is combined with one or more further anti-Rh(D) antibodies having one or more additional binding specificities. S

4. An anti-Rh(D) reagent as claimed in claim 3 wherein a monoclonal antibody capable of binding the D I variant is present.

5. An anti-Rh(D) reagent as claimed in claim 3 wherein the antibody component comprises: a monoclonal antibody as defined in claim 1, and an IgM anti-Rh(D) with no or only weak anti-Du activity. J A 32

6. An anti-Rh(D) reagent as claimed in claim wherein the antibody component additionally comprises an IgG monoclonal anti-Rh(D) capable of exhibiting activity against D vI cells in an indirect antiglobulin test.

7. An anti-Rh(D) reagent as claimed in claim 5 or claim 6 wherein component is selected from the monoclonal IgMs of the deposited hybridoma cell lines MAD-2 (ECACC 86041803) and FOM-l (ECACC 87021301).

8. A human lymphocyte-derived cell line capable of producing a monoclonal antibody as claimed in claim 1.

9. A cell line as claimed in claim 8 selected from 15 ECACC 87091605 and ECACC 87091604. A monoclonal antibody as claimed in claim 1 or claim 2 for use in passive immunisation to prevent haemolytic disease of the newborn. S 2 11. A culture supernatant obtained by cultivation of a cell line as claimed in claim 8 or claim 9.

12. A monoclonal antibody as claimed in claim 10 of the 25 allotype Glm(3).

13. A pharmaceutical composition for use in passive immunisation to prevent haemolytic disease of the newborn comprising a monoclonal antibody as claimed in claim 1 or claim 2 in association with a physio- logically acceptable carrier or diluent.

14. A pharmaceutical composition as claimed in claim 13 wherein said monoclonal antibody is of the allotype Glm(3) 9 33 A pharmaceutical composition as claimed in claim 13 or claim 14 wherein the antibody component additionally comprises a monoclonal antibody capable of binding the DVI variant.

16. A pharmaceutical composition as claimed in any one of claims 13 to 15 wherein the antibody component includes one or more anti-Rh(D) antibodies of the IgG3 sub-class.

17. A method of Rh-typing wherein a monoclonal antibody as claimed in claim 1 or claim 2 or an anti-Rh(D) reagent as claimed in any one of claims 3 to 7 is employed, said monoclonal antibody or said reagent being in the form of an aqueous solution. 71 j 1