B-cell Depletion in Offspring to Women With MS Under Immunomodulatory Treatment

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system affecting more women than men and typically manifests during reproductive ages (1). The disease and associated treatments therefore have a large impact on family planning. While trying to conceive most clinicians recommend discontinuation of disease-modifying drug use (DMD) before conception unless the risk of disease worsening outweighs the risk to the fetus (2,3). Patients with active disease may need to continue DMD treatment until conception or even during pregnancy. Furthermore, given that nearly half of all pregnancies are unplanned (4-6) women with MS may occasionally become pregnant during or shortly after receiving treatment. As such, gathering information on the effect of pregnancy exposure to DMD is crucial for the management of reproductive issues in women with MS.

Rituximab, a chimeric monoclonal B cell-depleting anti-CD20 antibody, is frequently used off-label for the treatment of MS in Sweden (7). The drug targets CD20 on the cell surface of B cells and effectively depletes them. Previously recommendations of rituximab was withdrawn at least one year prior to a planned pregnancy. Given the pharmacokinetics with a halftime of around 24 days current Swedish guidelines accept a minimum of 3-4 months between last infusion and conception. Rituximab is a chimeric antibody of the immunoglobulin G1 kappa type and is therefore actively transported over the blood-placenta barrier, mainly during the third trimester but starting already in the second trimester. Small case series and reports show that rituximab administered during the third trimester suppresses neonatal b-cell development (8-12). After six months, usually, b-cells levels normalize but effects may not be clinically apparent at birth but may influence response to vaccinations early in life and increase susceptibility to infections.

Severe combined immunodeficiency (SCID) is included in the Swedish national neonatal disease screening program, testing is performed at Centrum för Medfödda Metabola Sjukdomar (CMMS), in parallel with screening tests for other diseases, on blood samples in the form of dry blood spots (DBS) taken within 48 hours after birth. The SCID screening consists of assessing markers of T and B cell production, T-cell receptor excision circles (TREC) (13) and kappa-deleting recombination excision circles (KREC)(14) , respectively. Both are circular fragments of DNA that are excised during the gene rearrangements necessary for the formation of functional T- and B-cell receptors. KRECs are formed during the rearrangement of the kappa light chain gene and occurs in the bone marrow at the small pre-B-cell stage. CD20 is already expressed on the surface of these B-cell precursors and they may therefore be targeted by rituximab and other monoclonal anti-CD20 antibodies (15). Each cell forms one KREC copy, which by necessity is passed to only one of the cells after cell division. Consequently, a proportion of mature B-cells in the circulation will also carry KRECs, and because they express CD20 they may be depleted by rituximab.

We hypothesize that if rituximab inhibits B-cell formation in the fetus of a pregnant mother treated with the drug, this can be detected in the form of reduced KREC results. A case report that describes a child of a rituximab treated mother that had reduced KREC results at birth has been published suggesting that such effects occur (16). It has also been shown that broad immunosuppressants, in particular the purine analogue azathioprine, cause some KREC results that are under the cutoff in the neonatal screening in Sweden (17). More knowledge is needed regarding the safety of rituximab for the fetus during pregnancy to facilitate clinical decisions in this complex situation, being able to identify offspring with insufficient B-cell counts and consequently at higher risks for infection and influence of vaccination responds. Such knowledge will facilitate clinical decisions and may help identify offspring with insufficient B cell counts and consequently at higher risks for infection and poor vaccine response.

The experimental plan includes a retrospective part as well as a prospective part.

The retrospective part of the study will take advantage of already existing blood samples from the national newborn screening program and examine the KREC results of all children born to rituximab treated mothers with MS in Stockholm during the past 10 years. Information on DMD treatment, time of conception, B-cell level, immunoglobulin levels in the mothers at the end of each trimester are routinely collected and recorded in the Swedish Multiple Sclerosis registry (SMSreg). Mothers that have been treated with monoclonal CD-20 antibodies and other MS specific DMTs during pregnancy or within twelve months before conception and have successfully delivered a child will be included in the study. After informed consent has been received from the parents our collaborators at CMMS will extract KREC results if available from either the screening program or the preceding pilot study(17). If results are not available, the DBS samples will be retrieved and KREC analyzed specifically for this study. If the combined KREC results are ambiguous we have the option to retrieve DBS samples for all included children and perform an in-house assay developed at CMMS to confirm B-cell deficiency by determining abundance of CD19 transcripts in the sample. Furthermore, to be able to correlate B-cell deficiency to drug concentration in the child, DBS can be sent for drug level detection to our collaborators at the Haukeland University Hospital laboratory. The mothers B-cell deficiency and its consequence on immunoglobulin levels will also be assessed in the analysis.

In the prospective part of the study we plan to address if a reduction in the levels of KREC and/or CD19+ B-cells at birth of offspring to women with MS has clinical significance. Mothers with MS, regardless of treatment, that have successfully delivered or will deliver a child will be asked for informed consent included if applicable.

1. At clinical routine venous blood sampling immunoglobulin and B-cell levels are obtained. Additional samples, during clinical routine visit, in the becoming mother will allow analysis of drug concentration at the end of every trimester and 3 months post partum.
2. Within 1-12 months postpartum and after vaccination capillary blood samples from the child will be analyzed to detect antibodies induced by vaccination or infections to secure the ability to be able to develop antibodies despite being exposed to monoclonal CD20-antibodies in-utero or via breastmilk.
3. PKU-test is taken in the child within routine but additional analysis on drug levels and KREC will be analyzed as well as determining abundance of CD19 transcripts.
4. Within our routine contacts with the MS patient (mother) we will ask for infections in both, the mother and the child at 3 months and 1 year post-partum.

After informed consent has been received from the parents our collaborators at CMMS will also extract KREC results if available from the screening program. If results are not available, the DBS samples will be retrieved and KREC analyzed specifically for this study. If the combined KREC results are ambiguous we have the option to retrieve DBS samples for all included children and perform an in-house assay developed at CMMS to confirm B-cell deficiency by determining abundance of CD19 transcripts in the sample.

Primarily Rota virus vaccine will be considered because it is the earliest vaccination in the national vaccination program, offered at 6 weeks of age. Antibody levels for other vaccines or infections may be tested if deemed relevant. A detailed infectious and general medical history via the parents will be obtained at routine contacts between patient and health care and comparison of adverse outcomes between children with mothers exposed to rituximab and children to mothers with MS but without rituximab treatment within 12 months prior to conception as well as for children with and without apparent reduction in the levels of KREC and/or B-cell depletion due to in utero exposure to rituximab and children with normal KREC levels will be summarized.

In Canada, Germany and the US, breastfeeding despite receiving anti-CD20 antibody therapy is getting more common, assuming the antibody cannot be enterally absorbed hence eliminated fecally ungraded. Swedish recommendations at present are cessation of breastfeeding when starting DMT however more women choose to breastfeed regardless. We would like to offer a follow-up with breastmilk samples and blood samples in the child one month after drug application for drug level detection in breastmilk and child and B-cell level analysis in the child.

Sample analysis of B-cell levels, IgG and IgM levels as well as specific antibodies as detection for immune responds to vaccination will be analyzed with clinically established methods via the lab at Karolinska University Hospital and Lennart Svenssons lab in Linköping, Sweden. Blood sampling from the child will be performed at specialized blood sampling centers for children e.g. Karolinska university hospital. KRECs and abundance of CD19 transcripts will we analyzed in dried blood spots at CMMS with an established method within the clinics. As there is no existing established high-quality method in Sweden, we will have to send drug level analysis to the Bergen University in Norway, our collaborator. They will use ELISA and high-performance liquid chromatography-electrospray ionization mass spectrometry method to analyses drug levels in blood, breast milk and dried blood spots.

Descriptive data and differences in drug levels, cell levels, infection rate and KREC levels between women with MS who have been exposed to ant-CD20 antibodies and not close to their pregnancy and their offspring will be tested with chi2-tests or Wilcoxon rank sum test, where appropriate. IgG levels will be analyzed using a linear mixed effect model, including number of doses of administered DMT (i.e. RTX or other DMTs) as random effect, while age, sex, previous DMT, administered DMT and the interaction of doses and administered DMT as fixed effects. P-values \<0.05 will be considered statistically significant. Non-parametric variables will be presented as median (range), and normally distributed variables as mean (SD).

The dilemma of balancing the mothers need for effective treatment against potential harmful effects to the fetus is a common clinical problem. It is virtually impossible to study the effects of drugs on the developing child apart from gathering observational data from individual cases where, for any reason, the mother did receive a certain drug. In Sweden the conditions are especially good to gather information on the effects of in utero exposure to B-cell depleting drugs because the unique propensity to treat MS-patients with monoclonal CD20-antibodies (e.g. rituximab) in combination with a newborn screening program that includes a marker for B-cell deficiency. The study results will have direct impact on the clinical management of MS-patients with aggressive disease and family planning wishes.