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SYnAbs innovative monoclonal antibodies targeting hemoglobinopathia diseases

A short introduction to Hemoglobinopathia

 

Hemoglobinopathia are diseases associated with a genetic abnormality of hemoglobin, blood protein used to transport oxygen. Hemoglobin is found basically inside red blood cells (erythrocytes), which gives them their red color.

 

Human hemoglobin consists of four identical chains :

  • two α (alpha) chains
  • two β (beta) chains

Each of these channels is associated with a prosthetic grouping called heme. The name of hemoglobin comes from two words: heme and globin, symbolized by "Hb".

 

The alpha and beta genes of globin are coded respectively on the chromosomes 16 and 11. But like many proteins, chains hemoglobin present various mutations that have the most often no impact clinical. These diseases are autosomal recessive Mendelian transmission, meaning that the disease appears only in children whose both parents are carriers of the anomaly, it is the homozygous form. In the heterozygous form (only one affected parent), the disease is most often silent, remaining transmissible.

 

More than 500 hemoglobin abnormalities have been listed and they fall into two broad categories.

 

Of the importance of sickle cell disease diagnostic and newborn screening

 

More than 50 million people worldwide are affected by the Sickle cell disease, and more than 300'000 children with a serious form are born each year with the genetic anomaly. Sub-Saharan Africa is one of the prevalent area where we estimate 1 in every 13 newborns has developed the condition. Middle East, Carribean, India, Brazil are also main locations of Sickle cell disease.

 

Manifestations of the disease are multiple and can be listed as

  • "Vaso-occlusive" crises, caused by the obstruction of capillaries
  • Acute pain episodes due to vaso-occlusive seizures
  • Hand-foot or dactylitis syndrome
  • Hemolytic anemia
  • Acute chest syndrome
  • Stroke, Cerebrovascular accident
  • Increased susceptibility to infections
  • Chronic complications (Heart attack, kidney, dermatological, PAH, Retinopathy, Priapism, cholelithiasis…)

Since parents can be heterozygous for the disease, it is possible to have populations of asymptomatic healthy carriers of the disease. These adults can therefore be tested before conceiving a child to assess the risk of transmission to their offspring.

 

After conception, and if no screening has been done beforehand, early detection allows for early treatment and management, which can greatly improve the quality of life for those affected. Progress in the management of the disease has significantly increased the average life expectancy of people with sickle cell disease: it is now more than 40 years, whereas it was less than 20 years before the 1980s.

 

Treatments of sickle cell disease

 

So far, there is no cure for this genetic disease, except hematopoietic stem cell transplantation. Antibiotics, pain relievers and hydroxyurea are often used to help cope with the symptoms associated with the disease. Bluebird Bio is working on one-time treatment gene therapy approach with lentiviral vectors. New drugs like Voxelotor, crizanlizumab and PF-07055848 being developed by Global Blood Therapeutics, Novartis and Pfizer respectively, showed promising results in clinical trials.

 

 

Sickle cell disease diagnostic

 

Also called hemoglobinosis S, sicklemia or cell anemia falciformes, Sickle cell disease is a hereditary disease, autosomal recessive, which is characterized by an alteration of hemoglobin. The red blood cells of homozygous individuals, HbS / S, do not practically only contain HbS. These molecules have the property of polymerizing when deoxygenated, giving rise to training of fibers that deform the globule and give it an appearance in sickle" or crescent shape. 

 

The diagnosis is currently made:

  • by examining the shape of the red cells,
  • by analyzing hemoglobin by electrophoresis,
  • by genetic testing. 

Nevertheless, analyzing hemoglobin by electrophoresis presents certain limits:

  • It is not always able to detect all hemoglobin variants: some hemoglobin variants may not be separated by the standard electrophoresis conditions, making them difficult to detect.
  • It may not be able to distinguish between similar variants: some variants of hemoglobin may migrate at the same position on the gel, making it difficult to distinguish between them.
  • It may not detect low levels of hemoglobin variants: if the level of a specific hemoglobin variant is low, it may not be detected by electrophoresis, leading to false-negative results.
  • It takes time to get results: hemoglobin electrophoresis is typically done in a laboratory and it can take some time to get results, which may delay diagnosis and treatment.
  • It may not be as sensitive as other methods: Other methods such as HPLC or DNA-based methods may be more sensitive in detecting rare or low-level variants.

Regarding genetic testing for sickle cell disease, there are certain limitations :

  • False-negative results: genetic testing may not detect all mutations associated with SCD, particularly rare or unknown variants. This can lead to false-negative results, and further testing may be needed to confirm the diagnosis.
  • False-positive results: genetic testing may also yield false-positive results if an individual carries a genetic variant that is associated with SCD, but does not have the disease.
  • Limited diagnostic sensitivity: genetic testing for SCD may not detect all mutations, and it is not always possible to confirm a diagnosis based on genetic testing alone.
  • Cost and accessibility: genetic testing can be expensive and not all patients have access to genetic testing, particularly in low-income countries, with some geographical areas difficult to access and children not registered with the authorities.

Finally, examining red blood cell shape is a simple key diagnostic tool for sickle cell disease (SCD), as it allows identification of the characteristic sickle-shaped RBCs. However, there are certain limitations to this method:

  • Subjectivity: The assessment of RBC shape is often subjective and can be influenced by the skill and experience of the individual performing the analysis.
  • Variability: RBC shape can vary depending on the stage of the disease, making it difficult to accurately diagnose SCD in some cases.
  • Limited sensitivity: The examination of RBC shape may not detect all cases of SCD, particularly milder forms of the disease.
  • False-positive results: The examination of RBC shape may also yield false-positive results if an individual has RBCs that are abnormally shaped for reasons other than SCD.
  • It is not a quantitative measurement: It doesn't give a percentage of how many cells are sickled and it doesn't give information of the severity of the disease.

Sickle cell disease monoclonals

 

For all of the above reasons, SYnAbs has decided to develop a diagnostic tool with the following parameters:

  • High sensitivity: our monoclonal antibodies are highly specific and sensitive, which can help detect even low levels of abnormal hemoglobin in patients with sickle cell disease.
  • High specificity: our monoclonal antibodies are designed to specifically target each different format of hemoglobin variant, reducing the risk of false positive results.
  • Non-invasive
  • Easy to perform: our monoclonal antibodies can be used in a variety of diagnostic tests that are relatively simple to perform, such as Enzyme-linked Immunosorbent Assay (ELISA) but especially lateral flow immunoassays that can be performed in remote areas of urban environments in some developing countries
  • High throughput: these tests can be performed on a large number of samples at a time, making them a high-throughput technique.
  • Cost-effective: our monoclonal antibodies are produced in large quantities using different techniques from CELLines to roller bottles or bioreactor mode

SYnAbs has consequently developed monoclonal antibodies against different types of Sickle cells diseases, to produce sandwich ELISA and lateral flow assays. Thanks to its unique expertise to break immune tolerance to highly homologous compounds, coupled with rat monoclonal antibodies technologies, SYnAbs was able to target specific mutations on hemoglobin and generate immune response against very specific parts of these poor immunogenic antigens.

 

SYnAbs has now at your disposal off-the-shelf highly specific monoclonal antibodies to hemoglobin variants.

Anti-Hemoglobin A rat monoclonal antibodies : LO-HbA

 

Our anti-hemoglobin A rat monoclonal antibodies allow the specific detection of the most normal and current form of hemoglobin, without any cross-reaction to HbS in order to discriminate HbA homozygous patient from HBA-HbS heterozygous or HbS homozygous ones.

 

Anti-Hemoglobin C mouse monoclonal antibodies : MA-HbC

 

Our anti-hemoglobin C mouse monoclonal antibodies detect an abnormal hemoglobin in which glutamic acid residue at the 6th position of the β-globin chain is replaced with a lysine residue due to a point mutation in the HBB gene.

 

Anti-Hemoglobin E rat monoclonal antibodies : LO-HbE

 

Our anti-hemoglobin E rat monoclonal antibodies diagnose a single point mutation in the beta-globin gene (HBB) results in the substitution of a glutamic acid for lysine at the 26th position (Glu26Lys) in the beta chain. This genetic change causes the formation of a different hemoglobin called Hemoglobin E. Hemoglobin E (HbE) is the most common hemoglobin variant in Southeast Asia, particularly in Thailand, Cambodia, and Myanmar, where it is found in about 35% of the population. HbE is also found in other parts of the world, including India, Pakistan, and Bangladesh, but at a lower frequency. HbE is considered a benign variant and it is usually associated with a mild anemia and a decrease in red blood cells' oxygen-carrying capacity. However, in combination with another beta-globin gene mutation, such as beta-thalassemia, it can lead to a more severe form of anemia called HbE-beta thalassemia.

 

Anti-Hemoglobin Beta S rat monoclonal antibodies: LO-HbS

 

Our anti-hemoglobin S rat monoclonal antibodies detect a single point mutation in the beta-globin gene (HBB) results in the substitution of an amino acid valine for glutamic acid at the 6th position (Val6Glu) in the beta chain, without recognizing HbA.

 

Anti-pAN-Hemoglobin

 

Our anti-all hemoglobin mouse monoclonal antibodies can detect any kind of hemoglobin and such be used to sandwich format immuno-assay and have been positively tested on HbAA, HbSS, HbCC and HbEE.

 

Thalassemia monoclonals

 

Thalassemia are hereditary diseases, autosomal recessive are characterized by a lack of production hemoglobin :

  • Beta-thalassemia
  • Alpha-thalassemia

Beta-thalassemia affects mainly people from Mediterranean region, the Middle East, Asia (China, India, Vietnam, Thailand) and Black Africa. It reaches as many women than men. Around 200.000 people suffer from beta-thalassemia worldwide. Adult hemoglobinopathy testing market is growing by 3,9% (CAGR) and thalassemia market is estimated to reach 3,53$ billion by 2022.

 

According to the fact that the production of beta chains of globin is absent or only reduced, we distinguish:

  • ß-thalassemia major (Cooley anemia)
  • Intermediate ß-thalassemia
  • ß-thalassemia minor

ß-thalassemia major (Cooley anemia)

 

The first signs of ß-thalassemia major appear only after the age of 6 months because the newborn's blood still contains a lot of fetal hemoglobin HbF (alpha2 / gamma2), under a often very heterogeneous form according to individuals:

  • Severe hemolytic anemia
  • Fewer
  • Irritability
  • Hepatomegaly
  • Splenomegaly
  • Ictery
  • Developmental delay

To compensate for massive hemolysis, erythropoiesis is increased in bones leading to bone deformities. In the child the facial bones get thicker (deformation of the jaws,

flattening of the root of the nose, excessive spacing of the eyes).

 

Secondary complications due to iron overload following hemolysis / continuous transfusion may appear :

  • Endocrine and metabolic abnormalities
  • Hypogonadism 40-55%
  • Growth retardation 33%
  • Diabetes 6-13%
  • Hypothyroidism 10%
  • Heart complications
  • Cardiac insufficiency (hemosiderosis)
  • Arrhythmias
  • Cholelithiasis 

The diagnosis is currently performed :

  • Clinical suspicion (signs, symptoms, origin ...)
  • Blood smear (hypochromic anemia, microcytic)
  • Biochemistry: Hemolysis (free biliary, LDH, Haptoglobin)
  • Serum iron, ferritin
  • Electrophoresis of hemoglobin (HbA2 3.5-8% HbF 1-2%) 

Treatments are composed of :

o Blood transfusion

o Folic acid

o Iron Chelator

 

Intermediate ß-thalassemia

 

In intermediate beta-thalassemia, both beta genes are altered, but they still allow the manufacture hemoglobin in a reduced amount. The symptoms are therefore much less important than in Cooley anemia :

 

o Hypochromic microcytic anemia

o Bone abnormalities (+/-)

o No stunting

 

ß-thalassemia minor

 

Beta-thalassemia minor is due to mutation of only one of the two beta genes.

 

Generally, this form does not have consequence on health, since the other gene is able to compensate for the anomaly and make enough beta chains for produce a normal hemoglobin level or close to normal.

 

α-thalassemia

 

Alpha-thalassemia is very common. It mainly affects populations from Asia (Cambodia, Laos, Burma, Thailand in particular), in its intermediate or serious forms, from Africa equatorial, and the Mediterranean basin in its minor forms. Alpha-thalassemias occurs on chromosome 16, 2 genes, encode for the alpha strings of the globin.

 

The diagnosis is currently performed by:

  • Blood smear
  • Electrophoresis of hemoglobin