# Patent

## Title
Polypeptides capable of forming antigen binding structures with specificity for the Rhesus D antigens, the DNA encoding them and the process for their preparation and use

## Abstract

Polypeptides capable of forming antigen binding structures specific for Rhesus D antigens include the sequences indicated in the FIGS. 1a to 16b. The obtained polypeptides, being Fab fragments, MAY be used directly as an active ingredient in pharmaceutical and diagnostic compositions. The Fab and their DNA sequences can also be used for the preparation of complete recombinant Anti-Rhesus D antibodies. Useful in pharmaceutical and diagnostic compositions.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a Continuation of U.S. application Ser. No. 09/147,443, filed Jan. 21, 1999, incorporated herein by reference in its entirety, which is a National Stage of PCT/EP97/03253, filed Jun. 20, 1997, which claims priority to EPO 96810421.6, filed Jun. 24, 1996. The above applications are incorporated herein by reference in their entirety.


BACKGROUND OF THE INVENTION
This invention relates to polypeptides forming antigen binding structures with specificity for Rhesus D antigens and especially to Fab molecules with specificity for the Rhesus D antigen. The invention also relates to their application to provide pharmacological and diagnostic compositions. The above Fab fragments when genetically engineered to be part of complete antibodies are useful for the prophylaxis of hemolytic disease of the newborn (HDN). This invention provides the novel DNA and amino acid sequences of the above polypeptides.
Thus, the antibodies can be used for the protection of Rhesus negative women before or immediately after the birth of a Rhesus positive child to prevent HDN in subsequent pregnancies.
The invention also includes the application of the said Fab molecules either alone or in combination with Fc constant regions as complete antibodies for the purposes of treating other illnesses which might benefit from anti-Rhesus D immunoglobulin e.g. treatment of idiopathic thrombocytopenic purpura (ITP).
In addition anti-Rhesus D immunoglobulin can be used after mistransfusions of Rhesus positive blood to Rhesus negative recipients in order to prevent sensitization to the Rhesus D antigen. Further the invention relates to the application of these Fab fragments and antibodies as diagnostic reagents.
HDN is the general designation for hemolytic anemia of fetuses and newborn babies caused by antibodies of the mother. These antibodies are directed against antigens on the surface of the fetal erythrocytes. These antigens can belong to the Rhesus, ABO or other blood group systems.
The Rhesus blood group system includes 5 major antigens: D, C, c, E and e (Issitt, P. D., Med. Lab. Sci. 45:395, 1988). The D antigen is the most important of these antigens as it is highly immunogenic eliciting anti-Rhesus D antibodies during Rhesus incompatible pregnancies and following transfusion of Rhesus incompatible blood. The D antigen is found in approximately 85% of Caucasians in Europe and those individuals are said to be Rhesus positive, Individuals lacking the D antigen are called Rhesus negative. The expression of the D antigen can vary due to either low antigen density, hereafter known as weak D or Du, or due to partial antigenicity, hereafter known as partial D antigens.
The Rhesus D antigen, a membrane protein of the erythrocyte, has recently been cloned and its primary structure described (Le Van Kim, C., et al., PNAS 89:10925, 1992). Modeling studies suggest that the Rhesus D antigen has 12 transmembrane domains with only very short connecting regions extending outside the cell membrane or protruding into the cytoplasm.
The partial D phenotypes were first identified in people who carried D antigen on their red cells but who had an alloanti-D in their sera (Rose, R. R. and Sanger, R., Blood groups in man, Blackwell Scientific, Oxford, U.K. 1975; Tippett, P. et al., Vox Sanguinis. 70:123, 1996). This can be explained by regarding the D antigen as a mosaic structure with at least 9 different epitopes (epD1 to epD9). Thus in some D variant people the red cells lack part of this mosaic and antibodies are made to the missing D epitopes. Rhesus positive individuals that make antibodies against partial D antigens have been classified into six main different categories (DII to DVII) each having a different abnormality in the D antigen. More recently it has been shown that these D categories gave different patterns of reaction when tested against panels of human monoclonal anti-D antibodies (Tippett, P., et al., Vox Sanguinis. 70:123, 1996). The different reaction patterns identified the 9 epitopes and so define the different partial D categories. The number of epitopes present on the D antigen varies from one partial D category to another with the DVI category expressing the least, epD3, 4 and 9. The DVI category is clinically important as a DVI woman can be immunized strongly enough to cause hemolytic disease of the newborn.
The prophylactic efficacy of anti-RhD IgG for prevention of hemolytic disease of the newborn is well established and has been in routine use for many years. As a result this severe disease has become a rarity. Nevertheless the underlying cause of the disease, i.e. RhD incompatibility between a RhD negative mother carrying a RhD positive child still remains and thus requires a continual supply of therapeutic anti-RhD IgG.
In recent years the assurance of a continual supply of anti-RhD IgG has become an increasing problem. The pool of available hyperimmune serum from alloimmunized multiparous Rhesus negative women has drastically decreased due to the success of prophylactic anti-RhD. Thus the current methods of production require repeated immunization of an increasingly reluctant pool of donors for the production of high titer antiserum (Selinger, M., Br. J. Obstet. Gynaecol. 98:509, 1991). There are also associated risk factors and technical problems such as the use of Rhesus positive red blood cells for repeated immunization carrying the risk of transmission of viral diseases like hepatitis B, AIDS and other as yet unknown viruses (Hughes-Jones, N. C., Br. J. Haematol. 70:263, 1988). Therefore an alternative method for production of anti-RhD antibodies is required.
In the past few years various alternative sources of hyperimmune serum have been tried but all are associated with disadvantages. Epstein Barr Virus (EBV) transformation of lymphocytes creating B lymphoblastoid cell lines that secrete specific antibody including against the Rhesus D antigen (Crawford et al., Lancet. 386: Feb. 19th, 1983) are unstable and require extensive cloning. Also due to the low transformation efficiencies (1-3% of B cells) only a restricted range of antibody specificities can be obtained from the potential repertoire. Additionally it seems that mice do not respond to the Rhesus D antigen and thus no murine monoclonal antibodies are available which could be used for producing chimaeric or humanised antibodies. Until recently the only other alternative was production of human antibodies by the hybridoma technique which was also restricted by the lack of a suitable human myeloma cell fusion partner (Kozbor, D. and Roder, J. C., Immunol. Today. 4:72, 1983).
SUMMARY OF THE INVENTION
It is thus the object of the present invention to provide Fab fragments having a reactivity against the Rhesus D antigen as well as complete antibodies comprising the Fab fragments which are free from the above mentioned drawbacks.
In the last few years the technique of repertoire cloning and the construction of phage display libraries has opened up new possibilities to produce human antibodies of defined specificity (Williamson, R. A. et al., PNAS 90:4141, 1993). These methods were thus applied to the preparation of polypeptides capable of forming antigen binding structures with specificity for Rhesus D antigens, especially of Fab fragments having an activity against Rhesus D and partial D antigens.
The generation of human antibodies by repertoire cloning as described in recent years (Barbas III, C. F. and Lerner, R. A., Companion Methods Enzymol. 2:119, 1991) is based on isolating mRNA from peripheral B cells. This method offers the tools to isolate natural antibodies, autoantibodies or antibodies generated during the course of an immune response (Zebedee, S. L., et al., PNAS 89:3175, 1992; Vogel, M. et al., Eur. J. Immunol. 24:1200, 1994). This method relies on constructing a recombinant antibody library from a particular donor starting from the mRNA coding for immunoglobulin (Ig) molecules. As only the peripheral blood lymphocytes (PBL) can be isolated from a donor the chances of finding specific antibody producing B cells in the periphery are increased if an individual is boosted with the desired antigen shortly before harvesting the PBL (Persson, M. A. A., et al., PNAS 88:2432, 1991). The total RNA is then isolated and the mRNA of the Ig repertoire can be cloned using Ig specific primers in the polymerase chain reaction (PCR) followed by the co-expression of heavy and light chains of the Ig molecule on the surface of a filamentous phage particle thereby forming an “organism” that in analogy to a B cell can bind to an antigen. In the literature this method is also known as the combinatorial approach as it allows the independent combining of heavy and light chains to form a functional Fab antibody fragment attached to one of the tail proteins, called pIII, of a filamentous phage. Phages carrying the Fab molecules (hereafter known as Phab particles) are selected for the desired antigen specificity, by a process known as bio-panning. The antigen can be applied to a solid support, specific Phab bind to the antigen whilst non specific Phab are washed away and finally the specific Phab are eluted from the solid support. The specific Phab are then amplified in bacteria, allowed to re-bind to the antigen on the solid support and the whole process of bio-panning is repeated.
The successive rounds of panning and amplification of selected Phab in bacteria result in an enrichment of specific Phab that can be seen from a rise in titer of colony forming units (cfu) plated out after each round of panning. Our previous experience and published data indicate that specific phage can usually be detected after 4 to 6 panning rounds (Vogel, M. et al., Eur. J. Immunol. 24:1200, 1994). In the above cited related art there is, however, no hint that the indicated steps can be used for a successful preparation of Fab fragments of anti-Rh D antibodies.



BRIEF DESCRIPTION OF THE DRAWINGS
In the appended FIGS. la to 16b, DNA sequences coding for variable regions (V regions) of anti Rh D Fab fragments and the corresponding polypeptide sequences are disclosed as follows:
FIG. 1a is an LD1-40-VH sequence(SEQ ID NOS 1-2);
FIG. 1b is an LD1-40-VL sequence(SEQ ID NOS 3-4);
FIG. 2a is an LD1-52-VH sequence(SEQ ID NOS 5-6);
FIG. 2b is an LD1-52-VL sequence(SEQ ID NOS 7-8);
FIG. 3a is an LD1-84-VH sequence(SEQ ID NOS 9-10);
FIG. 3b is an LD1-84-VL sequence(SEQ ID NOS 11-12);
FIG. 4a is an LD1-110-VH sequence(SEQ ID NOS 13-14);
FIG. 4b is an LD1-110-VL sequence(SEQ ID NOS 15-16);
FIG. 5a is an LD1-117-VH sequence(SEQ ID NOS 17-18);
FIG. 5b is an LD1-117-VL sequence(SEQ ID NOS 19-20);
FIG. 6a is an LD2-1-VH sequence(SEQ ID NOS 21-22);
FIG. 6b is an LD2-1-VL sequence(SEQ ID NOS 23-24);
FIG. 7a is an LD2-4-VH sequence(SEQ ID NOS 25-26);
FIG. 7b is an LD2-4-VL sequence(SEQ ID NOS 27-28);
FIG. 8a is an LD2-5-VH sequence(SEQ ID NOS 29-30);
FIG. 8b is an LD2-5-VL sequence(SEQ ID NOS 31-32);
FIG. 9a is an LD2-10-VH sequence(SEQ ID NOS 33-34);
FIG. 9b is an LD2-10-VL sequence(SEQ ID NOS 35-36);
FIG. 10a is an LD2-11-VH sequence(SEQ ID NOS 37-38);
FIG. 10b is an LD2-11-VL sequence(SEQ ID NOS 39-40);
FIG. 11a is an LD2-14-VH sequence(SEQ ID NOS 41-42);
FIG. 11b is an LD2-14-VL sequence(SEQ ID NOS 43-44);
FIG. 12a is an LD2-17-VH sequence(SEQ ID NOS 45-46);
FIG. 12b is an LD2-17-VL sequence(SEQ ID NOS 47-48);
FIG. 13a is an LD2-20-VH sequence(SEQ ID NOS 49-50);
FIG. 13b is an LD2-20-VL sequence(SEQ ID NOS 51-52);
FIG. 14a is an LD1-6-17-VH sequence(SEQ ID NOS 53-54);
FIG. 14b is an LD1-6-17-VL sequence(SEQ ID NOS 55-56);
FIG. 15a is an LD1/2-6-3-VH sequence(SEQ ID NOS 57-58);
FIG. 15b is an LD1/2-6-3-VL sequence(SEQ ID NOS 59-60);
FIG. 16a is an LD1/2-6-33-VH sequence(SEQ ID NOS 61-62);
FIG. 16b is an LD1/2-6-33-VL sequence(SEQ ID NOS 63-64);
FIG. 17 shows the pComb3 expresion system;
FIG. 18 shows the preparation of genes of the heavy chains of the complete antibody; and
FIG. 19 shows the preparation fo genes of the light chains of the complete antibody.



DETAILED DESCRIPTION
In the appended FIGS. 1a to 16b; DNA sequences coding for variable regions (V regions) of anti Rh D Fab fragments and the corresponding polypeptide sequences are disclosed.
FIG. 17 shows a pComb3 expression system.
FIGS. 18 and 19 show the separate preparation of genes of the heavy and light chains of the complete antibody according to the description in example 6.
Subjects of the present invention are polypeptides capable of forming antigen binding structures with specificity for Rhesus D antigens according to the following definition:
Polypeptides capable of forming antigen binding structures with specificity for Rhesus D antigens which include Rhesus D-specific CDR 1, CDR 2 and CDR 3 regions of pairs of amino acid sequences VH and V L with the same or different identification numbers according to the figures given in the table below:














VH
VL















Identi-

CDR 1
CDR 2
CDR 3

CDR 1
CDR 2
CDR 3


fication

base pair
base pair
base pair

base pair
base pair
base pair


No.
Figure
No.
No.
No.
Figure
No.
No.
No.





LD1-40
FIG. 1a
91-105
148-198
295-342
FIG. 1b
64-96
142-162
259-288


LD1-52
FIG. 2a
91-105
148-198
295-342
FIG. 2b
64-96
142-162
259-288


LD1-84
FIG. 3a
91-105
148-198
295-342
FIG. 3b
64-96
142-162
259-285


LD1-110
FIG. 4a
91-105
148-198
295-342
FIG. 4b
64-96
142-162
259-285


LD1-117
FIG. 5a
91-105
148-198
295-345
FIG. 5b
64-96
142-162
259-288


LD2-1
FIG. 6a
91-105
148-198
295-342
FIG. 6b
61-99
145-165
262-294


LD2-4
FIG. 7a
91-105
148-198
295-342
FIG. 7b
64-96
142-162
259-282


LD2-5
FIG. 8a
91-105
148-198
295-342
FIG. 8b
64-96
142-162
259-288


LD2-10
FIG. 9a
91-105
148-198
298-345
FIG. 9b
 61-102
148-168
265-294


LD2-11
FIG. 10a
91-105
148-198
295-342
FIG. 10b
64-96
142-162
259-285


LD2-14
FIG. 11a
91-105
148-198
295-342
FIG. 11b
64-96
142-162
259-285


LD2-17
FIG. 12a
91-105
148-198
295-342
FIG. 12b
64-96
142-162
259-285


LD2-20
FIG. 13a
91-105
148-198
295-342
FIG. 13b
64-96
142-162
259-285


LD1-6-17
FIG. 14a
91-105
148-198
295-351
FIG. 14b
64-96
142-162
259-285


LD1/2-6-3
FIG. 15a
91-105
148-198
295-342
FIG. 15b
64-96
142-162
259-285


LD1/2-6-33
FIG. 16a
91-105
148-198
295-342
FIG. 16b
64-96
142-162
259-285









The identification number for each sequence is given. The locations of the Rhesus D specific CDR1 (complementarity determining region 1), CDR2 and CDR3 regions are indicated in the figures and according to base pair number in the table above. Preferred polypeptides according to the invention are anti-Rhesus D antibodies which include the variable regions of the heavy and light chains according to the sequences given in FIGS. 1a-16b. The FIGS. 1a, 2a, . . . 16a are related to the variable regions of the heavy chain and the FIGS. 1b, 2b, . . . 16b are related to the variable regions of the light chain.

Further subjects of the present invention are the DNA sequences coding for antigen binding polypeptides according to the following definition: DNA sequences coding for polypeptides capable of forming antigen binding structures with specificity for Rhesus D antigens which include regions with the Rhesus D-specific CDR 1, CDR2, and CDR3 segments of pairs of DNA sequences VH and VL with the same or different identification numbers according to the figures given in the table below and functional equivalents thereof:














VH
VL















Identi-

CDR 1
CDR 2
CDR 3

CDR 1
CDR 2
CDR 3


fication

base pair
base pair
base pair

base pair
base pair
base pair


No.
Figure
No.
No.
No.
Figure
No.
No.
No.





LD1-40
FIG. 1a
91-105
148-198
295-342
FIG. 1b
64-96
142-162
259-288


LD1-52
FIG. 2a
91-105
148-198
295-342
FIG. 2b
64-96
142-162
259-288


LD1-84
FIG. 3a
91-105
148-198
295-342
FIG. 3b
64-96
142-162
259-285


LD1-110
FIG. 4a
91-105
148-198
295-342
FIG. 4b
64-96
142-162
259-285


LD1-117
FIG. 5a
91-105
148-198
295-345
FIG. 5b
64-96
142-162
259-288


LD2-1
FIG. 6a
91-105
148-198
295-342
FIG. 6b
61-99
145-165
262-294


LD2-4
FIG. 7a
91-105
148-198
295-342
FIG. 7b
64-96
142-162
259-282


LD2-5
FIG. 8a
91-105
148-198
295-342
FIG. 8b
64-96
142-162
259-288


LD2-10
FIG. 9a
91-105
148-198
298-345
FIG. 9b
 61-102
148-168
265-294


LD2-11
FIG. 10a
91-105
148-198
295-342
FIG. 10b
64-96
142-162
259-285


LD2-14
FIG. 11a
91-105
148-198
295-342
FIG. 11b
64-96
142-162
259-285


LD2-17
FIG. 12a
91-105
148-198
295-342
FIG. 12b
64-96
142-162
259-285


LD2-20
FIG. 13a
91-105
148-198
295-342
FIG. 13b
64-96
142-162
259-285


LD1-6-17
FIG. 14a
91-105
148-198
295-351
FIG. 14b
64-96
142-162
259-285


LD1/2-6-3
FIG. 15a
91-105
148-198
295-342
FIG. 15b
64-96
142-162
259-285


LD1/2-6-33
FIG. 16a
91-105
148-198
295-342
FIG. 16b
64-96
142-162
259-285









Preferred DNA sequences are those coding for variable regions of Fab fragments of anti-Rh D antibodies according to the FIGS. 1a-16b. The FIGS. 1a, 2a, . . . 16a are related to the heavy chain and the FIGS. 1b, 2b, . . . 16b are related to the light chain.

A further subject of the present invention is a process for preparing recombinant Fab polypeptides according to the following definition:
A process for preparing recombinant polypeptides capable of forming antigen binding structures. e.g. Fab fragments, with specificity for Rhesus D antigens, which process comprises the following steps in sequential order:

    
    
        a) boosting an individual capable of forming anti-Rhesus D antibodies with Rhesus D positive red blood cells,
        b) isolating mononuclear cells from the individual,
        c) isolating total RNA from the mononuclear cells,
        d) preparing a cDNA by using an oligo(dT)primer. reverse transcribing the mRNA with M-MuLV reverse transcriptase, and amplifying the eDNA repertoire by a polymerase chain reaction using immunoglobulin gene family specific primers,
        e) creating a phage display library by inserting the DNA coding for the heavy and light chain of the Fab polypeptide into a phagemid vector, wherein the DNA for the heavy chain is inserted in frame to the gene coding for the phage protein pIII which allows the expression of a Fab pIII fusion protein on the surface of the phage,
        f) transforming bacterial cells with the obtained recombinant plasmids. cultivating the transformed bacterial cells, and co-expressing the heavy and the light chains of a Fab on filamentous phage particles,
        g) amplifying the Fab-carrying phage in bacteria, selecting individual phage clones by several rounds of panning on Rhesus positive red blood cells,
        i) isolating the plasmid DNA from the selected clones and cutting out the gill gene,
        j) transforming bacterial cells with the obtained plasmid. cultivating the transformed bacterial cells expressing the Fab. and isolating the Fab fragments.
    
    


A further subject of the present invention is a process for preparing recombinant polypeptides according to the following: A process for selecting recombinant polypeptides capable of forming antigen binding structures with specificity for Rhesus D antigens, and in particular showing reactivity with the Partial Rhesus DVI Variant and without any evidence of reactivity with red blood cells of Rhesus negative phenotypes, in particular without reactivity against the Rhesus alleles C, c, E, and e, which process comprises the following steps in sequential order:

    
    
        a) performing several negative absorptions on the following red blood cells: phenotype 1 (r′r, Ccddee) treated with bromelase, phenotype 1 not treated with bromelase, phenotype 2 (ryry, CCddEE) treated with bromelase and phenotype 2 not treated with bromelase,
        b) performing a positive absorption on DVI+red blood cells with or without bromelase treatment,
        c) determining the titer of phage binding to DVI+red blood cells,
        d )repeating steps a), b) and c) until the titer of phage binding to DVI+red blood cells has reached a satisfactory level.
    
    


Further subjects of the present invention are anti-Rh D antibodies according to the following definition: Anti-Rhesus D antibodies having heavy and light chain variable regions comprising the Rhesus D-specific CDR 1, CDR 2 and CDR 3 sequences of pairs of amino acid sequences VH VL having the same or different identification numbers according to the table below:














VH
VL















Identi-

CDR 1
CDR 2
CDR 3

CDR 1
CDR 2
CDR 3


fication

base pair
base pair
base pair

base pair
base pair
base pair


No.
Figure
No.
No.
No.
Figure
No.
No.
No.





LD1-40
FIG. 1a
91-105
148-198
295-342
FIG. 1b
64-96
142-162
259-288


LD1-52
FIG. 2a
91-105
148-198
295-342
FIG. 2b
64-96
142-162
259-288


LD1-84
FIG. 3a
91-105
148-198
295-342
FIG. 3b
64-96
142-162
259-285


LD1-110
FIG. 4a
91-105
148-198
295-342
FIG. 4b
64-96
142-162
259-285


LD1-117
FIG. 5a
91-105
148-198
295-345
FIG. 5b
64-96
142-162
259-288


LD2-1
FIG. 6a
91-105
148-198
295-342
FIG. 6b
61-99
145-165
262-294


LD2-4
FIG. 7a
91-105
148-198
295-342
FIG. 7b
64-96
142-162
259-282


LD2-5
FIG. 8a
91-105
148-198
295-342
FIG. 8b
64-96
142-162
259-288


LD2-10
FIG. 9a
91-105
148-198
298-345
FIG. 9b
 61-102
148-168
265-294


LD2-11
FIG. 10a
91-105
148-198
295-342
FIG. 10b
64-96
142-162
259-285


LD2-14
FIG. 11a
91-105
148-198
295-342
FIG. 11b
64-96
142-162
259-285


LD2-17
FIG. 12a
91-105
148-198
295-342
FIG. 12b
64-96
142-162
259-285


LD2-20
FIG. 13a
91-105
148-198
295-342
FIG. 13b
64-96
142-162
259-285


LD1-6-17
FIG. 14a
91-105
148-198
295-351
FIG. 14b
64-96
142-162
259-285


LD1/2-6-3
FIG. 15a
91-105
148-198
295-342
FIG. 15b
64-96
142-162
259-285


LD1/2-6-33
FIG. 16a
91-105
148-198
295-342
FIG. 16b
64-96
142-162
259-285









Preferably the antibodies are anti-Rh D immunoglobulin molecules comprising the heavy and light chain variable regions according to the FIGS. 1a to 16b combined with known heavy and light chain constant regions.

Further subjects of the present invention are pharmaceutical and diagnostic compositions comprising polypeptides, anti-Rh D antibodies or Fab fragments according to the invention.
The total re-amplified Phab population obtained after each panning can be tested for specificity using various methods such as ELISA and immunodot assays. It is also defined by the nature of the antigen e.g. anti-Rhesus D Phabs are detected by indirect haemagglutination using a rabbit anti-phage antibody or equivalent Coombs reagent as the cross linking antibody. Once a total Phab population has been identified as positive for the desired antigen, individual Phab clones are isolated and the DNA coding for the desired Fab molecules is sequenced. Individual Fab can then be produced by use of the pComb3 expression system which is illustrated in FIG. 16. In this system the gIII gene, coding for the tail protein pIII, is cut out from the phagemid vector pComb3. This allows production of soluble Fab in the bacterial periplasm. Such individual Fab fragments can then be tested for antigen specificity.
The phage display approach has also been used as a means of rescuing monoclonal antibodies from unstable hybridoma cell lines. This has been reported for anti-Rhesus D antibodies (Siegel, D. L. and Silberstein, L. E., Blood. 83:2334, 1994; Dziegiel, M. et al., J. Immunol. Methods. 182:7, 1995). A phage display library constructed from non-immunized donors has also been used to select Fv fragments (i.e. variable regions of heavy and light chains, VH and VL) specific for human blood group antigens which included one Fv fragment reacting against the Rhesus D antigen (Marks, J. D. et al., Biotechnology. 11:1145, 1993).
Important considerations when constructing combinatorial libraries are the source of cells used for RNA extraction and the nature of the antigen used for panning. Therefore, this invention uses a hyperimmune donor who was boosted i.v. with Rhesus D+ red blood cells (rbc). The PBL of the donor were harvested at +5 and +18 days after the i.v. boost and were used to construct 2 combinatorial libraries hereafter known as library D1 (LD1) and library D2 (LD2) respectively. Double immunofluorescence analysis of the harvested PBL, using the markers CD20 and CD38 for pan B cells and lymphoblastoid cells respectively, showed a higher than normal percentage of lymphoblastoid B cells, of plasma cell morphology. The high number of plasma cells found in the peripheral blood is most unusual as normally there are less than 1% in the periphery and probably indicates that the donor had a high percentage of circulating B cells with specificity for the Rhesus D antigen.
After construction of the library, the selection of Phabs specific for the Rhesus D antigen was achieved by bio-panning on fresh whole rbc of phenotype R1R1 (CDe/CDe) i.e. the reference cells used for Rhesus D typing. This was necessary since the Rhesus D antigen, an integral membrane protein of 417 amino acids (Le Van Kim, C. et al, PNAS 89:10925, 1992), loses its immunogenicity during purification (Paradis, G. et al, J. Immunol. 137:240, 1986) and therefore a chemically purified D antigen cannot be bound to a solid phase for selection of immunoreactive Phabs as for other antigen specificities previously selected in this system (Vogel, M. et al., Eur. J. Immunol. 24:1200, 1994). Modeling studies have suggested that only very short connecting regions of the Rhesus D antigen extend outside the cell membrane or protrude into the cytoplasm (Chérif-Zahar, B. et al, PNAS 87:6243, 1990). Thus the parts of the RhD antigen visible to antibodies are relatively restricted and may be under conformational constraint. This aspect of the Rhesus D antigen becomes even more important when considering selection of Phabs with reactivity against the partial D phenotypes which essentially lack certain defined epitopes of the D membrane protein (Mouro, I. et al, Blood. 83:1129, 1994).
Furthermore, since whole rbc do not only express the D antigen, a series of negative absorptions had to be performed on Rhesus D negative rbc in order to absorb out those Phabs reacting with the other antigenic proteins found on the rbc.
This panning procedure performed on Phabs coming from both LD1 and LD2 libraries resulted in the isolation of 6 different Fab producing clones from library LD1, 8 different Fab producing clones from library LD2 and 2 Fab producing clones from the pooled libraries LD1 and LD2.
The nomenclature and the figures where the sequences are listed are given in table 1.












TABLE 1





LIBRARY
VH-
VL-
LIBRARY
VH-
VL-


LD1
Sequence
Sequence
LD2
Sequence
Sequence


Clone No.
Figure
Figure
Clone No.
Figure
Figure







LD1-40
1a
1b
LD2-1
 6a
 6b


LD1-52
2a
2b
LD2-4
 7a
 7b


LD1-84
3a
3b
LD2-5
 8a
 8b


LD1-110
4a
4b
LD2-10
 9a
 9b


LD1-117
5a
5b
LD2-11
10a
10b





LD2-14
11a
11b





LD2-17
12a
12b





LD2-20
13a
13b









The above Fab clones show exclusive reactivity against the Rhesus D antigen, 3 of 5 Du rbc tested and agglutinating reactivity against the Partial D phenotypes as follows: Rh33, DIII, DIVa, DIVb, DVa, DVII.
However, using the above mentioned R1R1 rbc for panning of the Phabs, no clones were isolated which reacted against the Partial DVI phenotype. As the serum of the original hyperimmune donor tested at the time of construction of the recombinant library, was known to react against the DVI phenotype the recombinant library should also contain the anti-DVI specificity.
In order to select for the DVI reactivity the panning conditions were changed in that different cells were used. A special donor whose rbc had been typed and were known to express the Partial DVI phenotype was used as the source of cells for re-panning the LD1 and LD2 libraries. This second series of pannings was essentially performed in the same way as the first series except for the substitution of DVI rbc for R1R1 rbc and the addition of bromelase treatment to the DVI rbc. The DVI phenotype expresses the least number of Rhesus D epitopes and it is therefore difficult to make antibodies against it. It has been reported that only 15% of unselected polyclonal anti-D and 35% of selected anti-D made by Rhesus D negative subjects reacted with DVI+ cells (Mouro, I. et al, Blood. 83:1129, 1994). Bromelase treatment which removes N-acetylneuraminic acid (sialic acid) from the rbc membrane, was performed in order to render the Rhesus DVI epitopes more accessible during the panning with the pre-absorbed Phabs.
This second series of pannings on the LD1 library resulted in 1 Fab producing clone LD1-6-17. The nomenclature is given in table 2.











TABLE 2








VH-Sequence
VL-Sequence



LIBRARY LD1
figure
figure









Clone No: LD1-6-17
14a
14b










However this clone was reacting with Rhesus alleles C and E and showing a false positive reaction with DVI positive rbc. This was also due to the phenotype of the DVI donor (Cc DVI ee) who expressed the C allele which was not absorbed out by the Rhesus negative rbc (ccddee).
Thus a third series of pannings on a pool of the LD1 and LD2 libraries was performed using different rbc for the absorption phase. After 6 rounds of panning using both bromelase treated and non treated rbc for both the absorption steps and the elution from DVI positive rbc a total population of Phabs was obtained which reacted exclusively with rbc of phenotype R1R1 (CCDDee) and 2 different donors expressing the DVI variant.
This third series of pannings on the LD 1 and LD2 libraries resulted in 2 Fab producing clones reacting with DVI+ rbc. The nomenclature is given in table 3.











TABLE 3








VH-Sequence
VL-Sequence



LIBRARY LD1/LD2
figure
figure









Clone No: LD1/2-6-3
15a
15b



Clone No: LD1/2-6-33
16a
16b










Thus a total of 16 different anti-Rhesus D Fab clones have been isolated. The DNA from these clones has been isolated and sequenced using Fluorescent Cycle Sequencing on an ABI 373A Sequencing System. The nucleotide and corresponding amino acid sequences of the said Fab clones form the basis of this invention.
Sequence analysis has revealed that several clones were isolated bearing the same VH gene segment but different VL gene segments. This is the case for the two clones LD2-1 and LD2-10, for the two clones LD2-4 and LD2-11, and for the three clones LD2-14, LD1/2-6-3 and LD1/2-6-33, respectively.
The DNA sequences obtained and Fab fragments are useful for the preparation of complete antibodies having an activity against the Rhesus D antigen. Suitable expression systems for such antibodies are mouse myeloma cells or Chinese hamster ovary cells.
The examples which follow explain the invention in detail, without any restriction of the scope of the invention.
Example 1 describes the construction of 2 combinatorial libraries; especially the aforementioned LD1 and LD2 libraries.
Example 2 describes a series of pannings using R1R1 rbc on the said LD1 and LD2 libraries in detail.
Example 3 describes a series of pannings using both bromelase and non bromelase treated rbc for absorption and bromelase treated DVI positive rbc using a pool of the said LD1 and LD2 libraries.
Example 4 describes an indirect haemagglutination assay using a rabbit anti-phage antibody, as an equivalent Coombs reagent, to monitor the enrichment and specificity of Rhesus D specific Phabs after panning.
Example 5 describes the preparation and purification of Fab antibody fragments for application as diagnostic reagents.
Example 6 describes the preparation of complete anti-Rhesus D immunoglobulins using the sequences of the present invention.
EXAMPLE 1
Construction of the Recombinant LD1 and LD2 Libraries
a) Source of the Lymphocytes
A male adult who was a member of the volunteer pool of hyperimmune Rhesus D donors was given an i.v. boost of 2 ml of packed rbc from a known male donor of blood group O RhD+. The PBL were harvested at +5 and +18 days after the boost and the mononuclear cells (MNC) isolated by Ficoll gradient centrifugation (Lymphoprep, Pharmacia, Milwaukee, Wis.). The results of donor lymphocyte analysis of day +5 are given in table 4. The +5 day MNC were used directly for RNA preparation using a phenol-chloroform guanidinium isothiocyanate procedure (Chomczynski, P. and Sacchi, N., Anal. Biochem. 162:156, 1987). The +18 day MNC were first cultured for 3 days in RPMI-1640 medium (Seromed, Basel) containing 103 U/ml of IL-2 (Sandoz Research Center, Vienna, Austria) and 10 μg/ml of pokeweed mitogen (PWM; Sigma L9379, Buchs, Switzerland) before extracting RNA.







TABLE 4







Immunofluorescence analysis of donor


lymphocytes + 5 days after rbc i.v. boost












Cell

Cell




surface
% Positive
surface
% Positive



antigen
cells
antigen
cells
















CD20
15
CD8
12



CD38
20
CD25
7.6



CD20/38
15
CD57
12.5



CD3
47
CD14
6



CD4
34
HLA-DR
18










b) Construction of Library

Two separate libraries were constructed called LD 1 and LD2 (as detailed above) corresponding to the cells harvested at +5 days and +18 days (finally +21 days including the +3 days PWM stimulation) after the i.v. boost respectively. Total RNA was then prepared from these cells using a phenol-chloroform guanidinium isothiocyanate method. From this RNA, 10 μg were used to make cDNA using an oligo(dT) primer (400 ng) and reverse transcribed with M-MuLV reverse transcriptase according to the conditions specified by the supplier (Boehringer Mannheim Germany). PCR amplification was performed as described in Vogel, M. et al., E. J. of Immunol. 24:1200, 1994. Briefly, 100 μl PCR reaction contained Perkin-Elmer buffer with 10 mM MgCl2, 5 μl cDNA, 150 ng of each appropriate 5′ and 3′ primer, all four dNTP at 200 μM each and 2 U/ml Taq Polymerase (Perkin Elmer, NJ). The PCR amplification of the heavy and light chains of the Fab molecule was performed separately with a set of primers from Stratacyte (details given below). For the heavy chain six upstream primers were used that hybridize to each of the six families of the VH genes whereas one kappa and one lambda chain primer were used for the light chain. The downstream primers were designed to match the hinge region of the constant domains γ1 and γ3 for the heavy chain. For the light chain the downstream primers were matched to the 3′ end of kappa and lambda constant domains. The heavy and light chain PCR products were pooled separately, gel purified and cut with Xho1/Spe1 and Sac1/Xba1 restriction enzymes (Boehringer Mannheim), respectively. After digestion the PCR products were extracted once with phenol:chloroform:isoamylalcohol and purified by gel excision. The insertion of the Xho1/Spe1 digested Fd fragment and subsequent ligation of the Sac1/Xba1 digested light chain into the pComb3 vector, the transformation into XL1-Blue cells, and the production of phages were performed as described by (Barbas III, C. F. and Lerner, R. A., Companion Methods Enzymol. 2:119, 1991).
After transformation of the XL1-Blue E. coli cells samples were withdrawn and titrated on plates to determine the library size. These results indicated expression libraries of 7.5×106 and 7.7×106 cfu (colony forming units) for LD1 and LD2 respectively.
c) PCR Primers









VHI




5′-CAC TCC CAG GTG CAG CTG CTC GAG
(SEQ ID NO:65)


TCT GG-3′


 


VHII


5′-GTG CTG TCC CAG GTC AAC TTA CTC
(SEQ ID NO:66)


GAG TCT GG-3′


 


VHIII


5′-GTC CAG GTG GAG GTG CAG CTG CTC
(SEQ ID NO:67)


GAG TCT GG-3′


 


VHIV


5′-GTC CTG TCC CAG GTG CAG CTG CTC
(SEQ ID NO:68)


GAG TCG GG-3′


 


VHV


5′-GTC TGT GCC GAG GTG CAG CTG CTC
(SEQ ID NO:69)


GAG TCT GG-3′


 


VHVI


5′-GTC CTG TCA CAG GTA CAG CTG CTC
(SEQ ID NO:70)


GAG TCA GG-3′


 


CHI(gI)


5′-AGC ATC ACT AGT ACA AGA TTT GGG
(SEQ ID NO:71)


GTC-3′


 


VL(k)


5′-GT GCG AGA TGT GAG CTC GTG ATG
(SEQ ID NO:72)


ACC CAG TCT CCA-3′


 


CL(k)


5′-T CCT TCT AGA TTA CTA ACA CTC TCC
(SEQ ID NO:73)


CCT GTT GAA GCT CTT TGT GAC GGG CGA


ACT C-3′


 


VL(l)


5′G TGC ACA GGG TCC TGG GCC GAG CTC
(SEQ ID NO:74)


GTG GTG ACT CA-3′


 


CL(I)


5′G CAT TCT AGA CTA TTA TGA ACA TTC
(SEQ ID NO:75)


TGT AGG GGG-3′






d) Vectors and Bacterial Strains

The pComb3 vector used for cloning of the Fd and the light chain was obtained from the Scripps Research institute La Jolla, Calif.; (Barbas III, G. E. and Lerner, R. A., Companion Methods Enzymol. 2:119, 1991). The Escherichia coli strain XL1-Blue used for transformation of the pComb3 vector and the VCSM13 helper phage were purchased from Stratacyte (La Jolla, Calif.).
EXAMPLE 2
Selection of Rhesus D Phabs from LD1 and LD2 Libraries on R1R1 rbc
a) Absorption and B jo-Panning
A series of three negative absorptions on rbc group O Rh negative were performed for each panning round before positive selection on rbc group O Rh positive (R1R1). Fresh rbc were collected in ACD (acid citrate dextrose) anticoagulant and washed 3 times in 0.9% NaCl. The rbc were counted in Hayems solution and adjusted to 40×106/ml. Absorption: 1 ml of phage preparation in PBS/3% BSA was added to rbc group O Rh negative pellet (16×106 rbc) in 12 ml tubes (Greiner 187261, Reinach, Switzerland) and incubated at RT for 30 min. with careful shaking. All tubes were pre-blocked in PBS/3% BSA for a minimum of 1 hr at RT. The rbc were pelleted by centrifuging for 5 min. 300×g at 4° C. The resulting phage supernatant was carefully harvested and the process repeated twice more. After the final absorption the phage supernatant was added to the rbc group O Rh positive pellet (16×106 rbc) and again incubated at RT for 30 min. with gentle shaking. Then the rbc were washed at least 5 times in 10 ml ice cold PBS, centrifuged 5 min. 300×g at 4° C., followed by elution with 200 μl of 76 mM citric acid pH 2.8 for 6 min. at R.T. and neutralisation with 200 μl 1M Tris. The rbc were centrifuged 300×g, 5 min. at 4° C. and the resulting supernatant containing the eluted phages was carefully removed and stored with carrier protein (0.3% BSA) at 4° C. ready for re-amplification. The numbers of Rhesus D specific Phabs of each panning round are given in table 5.







TABLE 5







Selection of Rhesus D+ Phabs from


the LD1 and LD2 libraries on R1R1 rbc










No. of eluted Rhesus




D specific phages









Panning
Library D1
Library D2


Round No. a)
cfu
cfu





1
8 × 106
4.6 × 107


2
6 × 107
1.4 × 107


3
1 × 108
7.9 × 107


4
3 × 108
1.3 × 108


5
3 × 108
  1 × 108


6
nd
2.8 × 108





a) For each round 1012 Phabs were incubated in tubes with rbc Group O Rhesus negative (absorption phase) followed by elution from rbc Group O Rhesus positive (R1R1)


nd = not done


cfu = colony forming units






EXAMPLE 3
Selection of Rhesus D Phabs from the Pooled LD1 and LD2 Libraries on DVI+ rbc
a) Absorption on rbc Group O Rh Negative, Phenotypes 1 (r′r, Ccddee) and 2 (ryry, CCddEE)
A series of four negative absorptions on rbc group O Rh negative was performed for each panning round before positive selection on rbc group O Rh DVI positive. The negative absorptions were performed in the following order: Step 1) phenotype 1 treated with bromelase; step 2) phenotype 1 no bromelase; step 3) phenotype 2 treated with bromelase; step 4) phenotype 2 no bromelase. Frozen rbc were thawed into a mixture of sorbit and phosphate buffered saline, left standing in this solution for a minimum of 10 min. and then washed 5 to 6 times in phosphate buffered saline and finally stored in stabilising solution (DiaMed EC-Solution) ready for use. Before panning the rbc were washed 3 times in 0.9% NaCl. followed by counting in Hayems solution. Absorption: 1 ml of phage preparation in PBS/3% BSA was added to an rbc pellet (2×108) as in step 1 in 12 ml tubes (Greiner 187261, Reinach, Switzerland) and incubated at RT for 30 min. with careful shaking. All tubes were pre-blocked in PBS/3% BSA for a minimum of 1 hr at RT. The rbc were pelleted by centrifuging for 5 min. 300×g at 4° C. The resulting phage supernatant was carefully harvested and the process repeated using rbc as detailed above in steps 2, 3, and 4.
b) Treatment of rbc Rhesus D Negative r′r and ryry and Rhesus DVI+ with Bromelase
Bromelase 30 (Baxter, Düdingen, Switzerland) was used to treat rbc Rhesus DVI+ in the same proportions as used in a routine haemagglutination assay, i.e. 10 μl bromelase per 2×106 rbc. Thus bromelase was added to the required amount of rbc and incubated at 37° C. for 30 min. followed by washing 3 times in 0.9% NaCl, re-counting in Hayems solution and adjusting to the required concentration in PBS/3% BSA ready for Phab panning.
c) Bio-Panning on Bromelase Treated Rhesus DVI+ rbc
After the final absorption of rbc ryry non bromelase treated the phage supernatant was divided into 2 equal parts and added either to the enzyme or non enzyme treated rbc group O Rh DVI+ pellet (40×106) respectively and again incubated at RT for 30 min. with gentle shaking. Then the 2 populations of rbc were washed at least 5 times in 10 ml ice cold PBS, centrifuged 5 min. 300×g at 4° C., followed by elution with 200 μl of 76 mM citric acid pH 2.8 for 6 min. at R.T. and neutralisation with 200 μl 1M Tris. The rbc were centrifuged 300×g, 5 min. at 4° C. and the resulting supernatants containing the eluted phages from either the bromelase or non bromelase treated DVI+ rbc were carefully removed and stored with carrier protein (0.3% BSA) at 4° C. ready for re-amplification. In further rounds of panning the eluted phage from either the bromelase or non bromelase treated DVI+ rbc were kept separate and each followed the absorption protocol steps 1 to 4. The elution step was slightly different compared to panning round 1 as the phage populations were not again divided into 2 parts. Only those phage eluted from bromelase treated DVI+ rbc were also eluted again from bromelase treated DVI+ rbc and only those phage eluted from the non bromelase treated DVI+ rbc were also again eluted from non bromelase treated DVI+ rbc. The numbers of specific Phabs after each panning round are given in table 6.







TABLE 6







Selection of Rhesus D Phabs from pooled LD1 and LD2


libraries on Rhesus DVI+ red blood cells










No. of eluted Rhesus




DVI+ specific phages









Panning
−Bromelase
+Bromelase


Round No. a)
cfu
cfu





1
1.9 × 106
4.4 × 106  


2
1.6 × 106
4 × 105


3
2.4 × 107
4.1 × 107  


4
  3 × 106
5 × 107


5
  1 × 1078
1 × 108


6
nd
3 × 108





a) For each round 1012 Phabs were incubated in tubes with 2 different phenotypes of rbc Group O Rhesus negative (absorption phase) followed by elution from rbc Group O Rhesus DVI+.






EXAMPLE 4
Monitoring of the Panning Rounds and Determination of the Specificity of the Enriched Phabs Using a Rabbit Anti-Phage Antibody
Indirect Haemagglutination Assay
Freshly collected rbc of different ABO and Rhesus blood groups were washed 3 times in 0.9% NaCl and adjusted to a 3-5% solution (45-50×107/ml) in either 0.9% NaCl or PBS/3% BSA. For each test condition 50 μl rbc and 100 μl test (precipitated and amplified phage or control antibodies) were incubated together in glass blood grouping tubes (Baxter, Düdingen, Switzerland) for 30 min. at 37° C. The rbc were washed 3 times in 0.9% NaCl and then incubated with 2 drops of Coombs reagent (Baxter, Düdingen, Switzerland) for positive controls or with 100 μl of 1/1000 diluted rabbit anti-phage antibodies (made by immunising rabbits with phage VCSM13 preparation, followed by purification on an Affi-Gel Blue column and absorption on E. coli to remove E. coli-specific antibodies). The tubes were incubated for 20 min at 37° C., centrifuged 1 min at 125×g and rbc examined for agglutination by careful shaking and using a magnifier viewer.
When purified Fab were tested for agglutination, an affinity purified anti-Fab antibody (The Binding Site, Birmingham, U.K.) was used instead of the rabbit anti-phage antibody.
Table 7 shows the results of haemagglutination tests of Phab samples after different panning rounds on R1R1 rbc.
Table 8 shows the results of haemagglutination tests of Phab samples after different panning rounds on Rhesus DVI+ rbc.
Table 9 shows the reactivity pattern of individual Fab clones from libraries LD1 and LD2 with partial D variants.







TABLE 7







Monitoring of Phabs from LD1 and LD2 libraries by indirect


haemagglutination after panning on R1R1 rbc











Phab sample
Library LD1
Library LD2











Panning round
tested on rbc O Rh D+ (a)
















No. 4





undiluted
+
+



1⁄4
+
+/−



 1/20
−
−



No. 5



undiluted
++
+



1⁄4
++
+



 1/20
−
−



No. 6



undiluted
nd
+++



1⁄4
nd
++



 1/20
nd
nd



Helper phage (b)



undiluted, 1⁄4, 1/20
−
−







(a) Indirect haemagglutination was performed in glass tubes using 50 μl rbc (40 × 107/ml) and 100 μl Phabs starting at 4 × 1011/ml. After 30 min. at 37° C. the rbc were washed 3 times and further incubated for 20 min. at 37° C. with a 1/1000 dilution of rabbit anti-phage antibody.



(b) The M13 helper phage was used as a negative control and showed no nonspecific agglutination due to the phage particle alone. Agglutination was scored by visual assessment from +++ (strong agglutination) descending to − (no agglutination).



nd = not done













TABLE 8







Monitoring of Phabs from pooled LD1 and LD2 libraries by indirect


haemagglutination after panning on Rhesus DVI+ rbc








Phab sample
rbc phenotypes













Planning round
CCDDee
ccddee
Ccddee
CCddEE
DVI (E.J.)
DVI (K.S.)





Non Bromelase








treated rbc DVI+


Round No. 3
a) +++ 
−
+/−
(+)
+/−
+/−


Round No. 5
++
−
−
−
−
−


Bromelase


treated rbc DVI+


Round No. 4
+++
−
+/−
−
(+)
+/−


Round No. 5
+++
−
+/−
+/−
(+++)
++


Round No. 6
++++
−
−
−
+++
+++








LD1 - 6 - 17
reactive with C and E













LD1/2 - 6 - 3
++++
−
−
−
+/−
nd


LD1/2 - 6 - 33
++++
−
−
−
+
nd





a) Agglutination was scored by visual assessment from ++++ (strong agglutination) descending to − (no agglutination).


nd = not done


Note:


Only those Phabs eluted from bromelase trated DVI+ rbc showed evidence of agglutination against 2 different DVI+ donors.













TABLE 9







Clonal Analysis of Reactivity of Fab anti-Rhesus D Clones


from Libraries D1 and LD2 against Partial D Variants









Partial D Variants














(a) Fab Clone No
RH33
DIII
DIVa
DIVb
DVa
DVI
DVII


















LD1
- 40
−
b) +++  
+
+
+/−
−
++



- 52
−
+++
−
−
+++
−
+++



- 84
−
++
−
−
−
−
+



- 110
(+)
+++
++
+
+
−
++



- 117
−
+++
−
−
−
−
++


LD2
- 1
+++
nd
+++
+++
+
−
+++



- 4
−
+++
−
+
−
−
+



- 5
−
Nd
+++
+++
−
−
+++



- 10
(−)
+++
+++
+++
+
−
++



- 11
−
+++
−
−
−
−
++



- 14
+++
+++
+++
+++
+++
−
+++



- 17
−
+++
+++
+
+/−
−
+++



- 20
−
+++
+++
−
+/−
−
+++














LD1/2-6-33
++
+++
+++
++
+++
+
++


LD1/2-6-33
+/−
+++
+++
++
+++
+
++





(a) soluble Fab preparations were made of each clone followed by indirect haemagglutination.


b) Agglutination was scored by visual assessment from +++ (all cells agglutinated in a clump) descending to − (no cells agglutinated).






EXAMPLE 5
Preparation and Purification of Fab Antibody Fragments for Application as Diagnostic Reagents
After the bio-panning procedures detailed in Examples 2 and 3 a phage population which showed specific agglutination on Rhesus D+ rbc was selected and used to prepare phagemid DNA. More precisely the Phabs selected on R1R1 rbc were used after the 5th and 6th rounds of bio-panning for LD1 and LD2 libraries respectively and after the 5th bio-panning on DVI+ rbc for isolation of the LD1-6-17 clone, in order to produce soluble Fab, the sequence gIII coding for the pIII tail protein of the phage particle must be deleted.
Phagemid DNA was prepared using a Nucleotrap kit (Machery-Nagel) and the gIII sequence was removed by digesting the so isolated phagemid DNA with Nhe1/Spe1 as described (Burton, D. R., et al., PNAS, 1989). After transformation into XL1-Blue individual clones were selected (nomenclature given in table 1) and grown in LB (Luria Broth) containing 50 μg/ml carbenicillin at 37° C. to an OD of 0.6 at 600 nm. Cultures were induced with 2 mM isopropyl β-D-thiogalactopyranoside (IPTG) (Biofinex, Praroman, Switzerland) and grown overnight at 37° C. The whole culture was spun at 10,000×g for 30 min. at 4° C. to pellet the bacteria. The bacterial pellet was treated with a lysozyme/DNase solution to liberate the Fab fragments inside the cells. As some Fab were released into the culture supernatant this was also harvested separately. These Fab preparations were then pooled and precipitated with 60% ammonium sulphate (Merck, Darmstadt, Germany) to concentrate the Fab followed by extensive dialysis in phosphate buffered saline (PBS) and ultracentrifugation at 200,000×g to pellet any insoluble complexes. The Fab preparations were then purified on a ceramic hydroxyapatite column (HTP Econo cartridge, BioRad, Glattbrugg, Switzerland) using a gradient elution of PBS (Buffer A) and PBS+0.5M NaCl (Buffer B). The linear gradient was programmed to increase from 0-100% Buffer B in 40 min. The Fab was eluted as a single peak between 40-60% Buffer B. The positive fractions as identified by immunodot assay using an anti-Fab peroxidase conjugate (The Binding Site, Birmingham, U.K.) were pooled, concentrated using polyethylene glycol and extensively dialysed against PBS. The positive fractions from the hydroxyapatite column for each clone were used in a classical indirect haemagglutination assay in glass tubes using either the standard Coombs reagent (Baxter Diagnostics AG Dade, anti-human serum) or an anti-Fab (The Binding Site, Birmingham, U.K.) as the cross linking reagent. These Fab of defined specificity on the Partial D variants as shown in Table 9 can be used to type rbc of unknown Partial D phenotype.
EXAMPLE 6
Construction of Complete Immunoglobulin Genes
The LD2-14 heavy chain V gene (VH gene) was amplified from the anti-Rhesus D-Fab-encoding plasmid LD2-14 with the polymerase chain reaction (PCR) using specific primers. The 5′-primer had the sequence:








(SEQ ID NO:78)











5′-GGGTCGACGCACAGGTGAAACTGCTCGAGTCTGG-3′







whereas the 3′-primer was of the sequence:









(SEQ ID NO:79)











5′-GCCGATGTGTAAGGTGACCGTGGTCCCCTTG-3′







The PCR reaction was performed with Deep Vent DNA Polymerase and the buffer solution (2 mM Mg++) from New England Biolabs at the conditions recommended by the manufacturer including 100 pmol of each primer and the four deoxynucleotides at a concentration of 250 μM each. The reaction was run for 30 cycles with the following temperature steps: 60 s at 94° C. (extended by 2 min. during the first cycle), 60 s at 57° C. and 60 s at 72° C. (extended by 10 min. during the last cycle). Post-amplification addition of 3′ A-overhangs was accomplished by a subsequent incubation for 10 min at 72° C. in the presence of 1 unit Taq DNA Polymerase (Boehringer Mannheim, Germany). The PCR product was purified using the QIAquick PCR purification kit (Qiagen, Switzerland) and cloned into the vector pCRII using Invitrogen'S TA cloning kit (San Diego, USA). Having digested the resulting plasmid TAVH14 with SalI and BstEII, the VH gene was isolated by preparative agarose gel electrophoresis using Qiagen's QIAquick gel extraction kit.
Vector #150 (Sandoz Pharma, Basel) which contained an irrelevant but intact human genomic immunoglobulin VH gene was cut with SalI and BstEII, and the vector fragment was isolated by preparative agarose gel electrophoresis using Qiagen's QIAquick gel extraction kit. Ligation of vector and PCR product was performed at 25° C. for 2 hours in a total volume of 20 μl using the rapid DNA Ligation kit (Boehringer Mannheim, Germany). Following ligation, the reaction mix was diluted with 20 μl H2O and extracted with 10 volumes of n-butanol to remove salts. The DNA was then pelleted by centrifugation, vacuum dried and resuspended in 10 μl H2O. 5 μl of this DNA solution were electroporated (0.1 cm cuvettes, 1.9 kV, 200Ω, 25 μFD) with a GenePulser (BioRad, Gaithersburg) into 40 μl of electroporation competent E. coli XL1-blue MRF′ (Stratagene, La Jolla), diluted with SOC medium, incubated at 37° C. for 1 hour and plated on LB plates containing ampicillin (50 μg/ml). Plasmid-minipreps (Qiagen, Basel) of the resulting colonies were checked with restriction digests for the presence of the appropriate insert.
With this procedure, the irrelevant resident VH gene in vector #150 was replaced by the amplified anti-Rhesus D VH sequence of LD2-14 and yielded plasmid cassVH14. The structure of the resulting immunoglobulin VH gene construct was confirmed by sequencing, cut out by digestion with EcoRI and BamHI and gel purified as described above. Expression vector # 10 (Sandoz Pharma, Basel) containing the human genomic immunoglobulin Cγ1 gene segment was also digested with EcoRI and BamHI, isolated by preparative agarose gel electrophoresis, ligated with the EcoRI/BamHI-VH gene segment previously obtained from plasmid cassVH14 and electroporated into E. coli XL1-blue MRF′ as outlined above. This resulted in a complete anti-Rhesus D heavy chain immunoglobulin gene in the expression vector 14IgG1(Fig.18).
The LD2-14 light chain V gene (VL gene) was amplified from the same anti-Rhesus D-Fab plasmid LD2-14 by PCR using specific primers. The 5′-primer had the sequence:








(SEQ ID NO:76)











5′-TACGCGTTGTGACATCGTGATGACCCAGTCTCCAT-3′







whereas the 3′-primer was of the sequence:









(SEQ ID NO:77)











5′-AGTCGCTCAGTTCGTTTGATTTCAAGCTTGGTCC-3′







PCR reaction, product purification and subsequent cloning steps were analogous to the steps described for the VH gene, except that the appropriate light chain vectors were used. Briefly, the VL PCR product was cloned into pCRII vector yielding plasmid TAVL14, excised therefrom with MluI and HindIII and isolated by gel extraction. The VL gene was subsequently cloned into the MluI and HindIII sites of vector # 151 (Sandoz Pharma, Basel) thus replacing the irrelevant resident VL gene by the amplified anti-Rhesus D VL sequence of LD2-14. Having confirmed the sequence of the resulting plasmid cassVL-14, the EcoRI/XbaI fragment containing the VL gene was then subcloned into the restriction sites EcoRI and XbaI of vector # 98 (Sandoz Pharma, Basel, Switzerland) which contains the human genomic immunoglobulin Cκ gene segment. This procedure replaced the irrelevant resident VL gene in plasmid # 98 and yielded the expression vector 14kappa which contains the complete anti-Rhesus D light chain immunoglobulin gene.
The mouse myeloma cell line SP2/0-Ag 14 (ATCC CRL 1581) was cotransfected by electroporation with the expression vectors 14IgG1 and 14kappa previously linearized at the unique EcoRI and NotI cleavage site, respectively. The electroporation was performed as follows: exponentially growing cells were washed twice and suspended in phosphate buffered sucrose (272 mM sucrose, 1 mM MgCl2, 7 mM NaH2PO4, pH 7.4) at a density of 2×107 cells/ml. 0.8 ml of cells were added to a 0.4 cm cuvette, mixed with 15 μg of linearized plasmids 14IgG1 and 14kappa, held on ice for 15 min., electroporated with 290 Volts, 200Ω, 25 μFD, put back on ice for 15 min., transferred to a T75 cell culture flask with 20 ml of cold RPMI 1640 medium (10% heat inactivated fetal bovine serum, 50 μM beta-mercaptoethanol), left for 2 h at room temperature and then incubated for 60 h at 37° C. After this period, the cells were transferred to 50 ml of medium containing 1 mg/ml G418 for selection. Stable transfectants were then selected in the presence of increasing concentrations of methotrexate to amplify the integrated DNA and thus increasing the expression of the corresponding antibody rD2-14.
Expression of rD2-14 in the culture's supernatant (SrD2-14) was monitored by an enzyme linked immuno-sorbent assay (ELISA) specific for human γ1 and kappa chains. Quantification of the Rhesus D specific immunoglobulins in the anti-D assay according to Ph. Eur. revealed between 1.1 and 11.4 μg/ml of agglutinating antibody in such supernatants. They tested agglutination negative for Rhesus negative rbc and revealed the same agglutination potential against partial D variants as the Fab LD2-14 expressed in E. coli. The data are shown in table 10.







TABLE 10







Comparative analysis of reactivity of Fab anti-Rhesus D clone


LD2-14 and antibody rD2-14 against partial D variants









Partial D Variants

















R1R1
rr
Rh33
DIII
DIVa
DIVb
DVa
DVI
DVII




















LD2-14
+++
−
+++
+++
+++
+++
+++
−
+++


SrD2-14
+++
−
+++
+++
+++
+++
+++
−
+++


TCB
−
−





Agglutination was scored by visual assessment from +++ (all cells agglutinated in a clump) descending to − (no cells agglutinated).


LD2-14: Fab fragment prepared as described in Example 5;


SrD2-14: cell culture supernatant containing antibody rD2-14;


TCB: cell culture supernatant of untransfected cells.

## Claims

1. A purified polypeptide capable of forming antigen binding structures with specificity for Rhesus D antigens of the partial DVI phenotype comprising
a VH region having SEQ ID NO: 58 or SEQ ID NO: 62, and
a VL region.

2. The polypeptide of claim 1, wherein the polypeptide is an antigen binding Fab fragment.

3. The polypeptide of claim 1, wherein the polypeptide is an immunoglobulin specific for a Rhesus D antigen.

4. The polypeptide of claim 3, wherein the immunoglobulin comprises at least one defined isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

5. A recombinant polynucleotide which encodes the polypeptide of claim 1.

6. A composition comprising at least one polypeptide of claim 1 and a pharmaceutically acceptable carrier.

7. A composition comprising at least one immunoglobulin of claim 3 and a pharmaceutically acceptable carrier.

8. A diagnostic composition for Rhesus D typing comprising at least one polypeptide of claim 1.

9. A diagnostic composition for Rhesus D typing comprising at least one immunoglobulin of claim 3.