Human antibodies for the generation of new red blood cell antigen typing reagents.

Monoclonal antibodies are used for red blood cell (RBC) antigen typing of
patients and donors, routinely by using agglutination-based assays. Correct
typing is of utmost importance for safe blood transfusions. For rare blood
group antigens, only limited antisera are available, and some of them are not
reliable. In other cases, available typing reagents are of the IgG type while
IgG antibodies are not functionally adequate. IgG antibodies do not lead to
good agglutination and to compensate for that a Coombs reagent needs to be
applied (anti-IgG) to enhance the agglutination. These problems can be
circumvented when the testing reagent is an *anti-blood group* antibody of the
IgM isotype, which can be used directly to induce agglutination. Most
importantly, current supplies of available antibodies against blood groups will
become unavailable in 3-5 years. There is therefore a compelling need to
generate a new and unlimited supply of monoclonal antibodies that can be used
for RBC antigen typing.

Solve above mentioned problems by cloning new and existing IgG-based
monoclonals for repurpose them as recombinant directly agglutinating IgM typing
reagents.

When at Sanquin Immunohematology Diagnostics an antibody of interest is
detected in a patient, the treating physician will be contacted to ask the
patient if the researcher is allowed to ask the patient for participation.
After approval, the Sanquin Researcher will contact the patient for
participation in the research. After informed consent (see F1) is obtained a
one-time blood sample of 36 ml will be taken. Considering the patient's
preference, sampling will be done at a Sanquin location or by the general
practitioner. In case of the latter a package with all necessities for blood
sampling will be sent to the patient.

The antibodies of interest (not exclusievly) are anti-Fya, -Fyb, -Kpa, -Kpb,
-Lua, -Lub, -Xga, -Wra, -Cob and -s.

From the patients* mononuclear cells, all CD19+ B cells are purified. To enrich
for antibody-specific B cells, antigen specific erythrocyte ghosts are added to
CD19+ B cells and all antibody-specific B cell-ghost cell complexes are sorted
as single cells in 96-well flat-bottom plates. No viable cells of the patient
will be stored. The B cells are cultured for 7 days, after which supernatants
are harvested and tested for agglutination with a panel of antigen positive and
negative RBC. From the progeny of the cells that produced the antibody, RNA is
extracted and reverse-transcribed. During this RNA extraction procedure, the
cultured patient cells are lysed, so no viable patient cells will be left. The
cDNA is amplified and PCR products are sequenced and cloned into vectors
containing the variable kappa and each of the four human IgM or IgG subclasses
constant regions. HEK293 (Human Embryonic Kidney) cells are transfected with
DNA, cultured for 5 days and IgG and IgM antibodies are purified from the
supernatant. These recombinant vectors can be multiplied to generate an
unlimited supply of the antibodies. The original plasma of the patient
containing the polyclonal alloantibodies will be stored, to compare reactivity
with the recombinant antibodies.

As a proof of concept, we recently successfully applied these techniques to
produce a unique anti-Vel-specific IgM class antibody, that was able to
distinguish between Vel-negative and very weak Vel antigen expressing RBC by
direct agglutination
(reference: van der Rijst MVE, Lissenberg-Thunnissen SN, Ligthart PC, Visser R,
Jongerius JM, Voorn L, Veldhuisen B, Vidarsson G, van den Akker E, van der
Schoot CE. Development of a recombinant anti-Vel immunoglobulin M to identify
Vel-negative donors. Transfusion. 2019;59:1359-1366).

Patients will undergo a one time venipuncture for the collection of 36 ml blood.
A venipuncture is regarded an intervention with minimal risks