Biopharm pdts: cytokines and Ab therapy

Question 1

Can other cell types besides immune cells secrete cytokines?

Answer 1

Yes, e.g., fibroblast, epithelial cells, adipocytes

Question 2

Cytokines are secreted to mediate __, __, and __

Answer 2

immunity, inflammation, and hematopoiesis

Question 3

Describe cytokine action

Answer 3

Bind to cell surface membrane receptors of effector cells, trigger intracellular signaling events, alter gene expression in target cells, induce changes in protein synthesis

Question 4

What are 2 types of cytokine therapeutics?

Answer 4

1. Recombinant Cytokines (activation)
   - incr receptor activation, boost immunity
2. Anti-cytokine (suppression)
   - mAb bind to cytokine ligand, prevent cytokine from binding to the receptor, no activation

Question 5

Describe the action of Interferons (IFNs)

Type 1 interferons (IFNa, IFNB)
Type 2 inteferons (IFNy)

Answer 5

Broadly, interferons are produced by virally infected cells, to help promote “antiviral state” (but mostly just IFNa with antiviral action)

IFNa: produced by leukocytes, enhance antiviral actions by increasing transcription and expression of MHC molecules, to activate T cells

IFNB: produced by somatic cells such as fibroblasts and epithelial cells, used in tx of multiple sclerosis ==> inhibit IFNy activity, slow growth of attacking immune cells, stops pdn of myelin-destroying compounds

IFNy: produced by T lymphocytes
- activate resting macrophages + monocytes to incr their phagocytic activity
- induce macrophages to express cytokines (IL2, TNFa), MHC proteins, Fc receptors

Question 6

Describe the action of interleukins (IL)
IL2 and IL11

Answer 6

Broadly affects growth, differentiation, and activation of immune cells and hematopoeitic (thrombopoietic) cells

IL2: produced by T cells, stimulates growth, differentiation, and activation of T cells, B cells, and NK cells

IL11: thrombopoietic growth factor produced by fibroblast and bone marrow stromal cells, stimulated proliferation of hematopoietic stem cells and induces megakaryocyte maturation (incr platelet formation)

Question 7

Describe the action of hematopoietic growth factors
List some examples

Answer 7

Stimulate differentiation of blood cells from bone marrow precursors

Promote hematopoiesis from pluripotent hematopoietic stem cells to functional immunologically active cells

E.g.,
Granulocyte-colony stimulating factor (G-CSF, Grastim, can incr neutrophil counts),
GM-CSF (Gramostim, incr neutrophils, eosinophils, monocytes)
Erythropoietin (EPO, incr RBC counts),
Some interleukins (e.g., IL11, incr platelet counts)

Question 8

Describe the action of chemokines

Answer 8

Stimulate leukocyte chemotaxis (migration), to attract immune cells to the site of inflammation

Question 9

Describe action of TNF and adipokine

Hint: the “bad” cytokines, may be in response to inflammatory conditions (cytokine storm)

Answer 9

TNF: pro-inflammatory, pro-apoptosis

Adipokine: produced by adipocytes, contribute to insulin resistance

Question 10

Name the 5 classes of cytokines

Answer 10

1. Interferons
2. Interleukins
3. Colony stimulating factors
4. Chemokines
5. Others (TNF, adipokine)

Question 11

Describe the production process of antiserum (polyclonal antibodies)
(from immunized animal - e.g., rabbit anti-mouse insulin polyclonal Abs)

Answer 11

1. Whole blood from immunized animal collected and left to clot/add coagulant
2. Clotting factors removed
3. Serum obtained as supernatent after centrifugation

Question 12

How is raw serum w polyclonal antibodies purified? (There are 2 methods)

Answer 12

1. Protein A/G purification
   - Protein A/G have affinity for Fc domain of Ig
   - Protein A/G placed on stationary phase (resin beads) of column chromatography
2. Immunoaffinity column chromatography
   - Antigen covalently bound to column (stationary phase), retain Ig of desired specificity

Question 13

When is antiserum (polyclonal antibodies) used?

Answer 13

For passive immunization - patient receive antibody instead of making own

E.g., snake venom antisera, botulism antitoxin
==> These are rare, antiserum with polyclonal antibodies would be cheaper tx option

Question 14

What are the 3 limitations of murine (mouse) monoclonal antibodies?
What is the naming convention for murine mAbs?

Answer 14

-omab

1. May induce immunogenicity (e.g., human anti-mouse antibody response - joint swelling, rashes, kidney failure)
2. Fail to trigger some effector functions
3. Short half life

Question 15

What is retained (not humanized) in chimeric monoclonal antibodies?
What is the naming convention for chimeric mAbs?

Answer 15

-ximab

75% human, only the AA sequences on VH and VL regions are conserved to retain similar antigen selectivity and affinity

AA sequences on CH and CL regions are replaced

Question 16

What is retained (not humanized) in humanized monoclonal antibodies?
What is the naming convention for humanized mAbs?

Answer 16

-zumab

> 90% human, only the hypervariable CDR of VH and VL domains are retained
12 CDRs retained in 1 antibody monomer

Since CDR responsible for antigen specificity and affinity, only retain this

Question 17

Recombinant human mAb uses recombinant DNA technology to genetically engineer mammalian host cells. How is this done?
What is the naming convention for recombinant human mAbs?

Answer 17

-umab / -mumab

Vectors are used to clone genes that encode the light and heavy chains of recombinant mAbs.

Question 18

Explain the structure and action of naturally derived antibody derivative, Ig conjugate

Answer 18

2 Fab arms and 1 Fc domain still present

Fc domain is conjugated with radioisotope/cytokine/toxin
Antibody binds to surface molecule of a cell to form surface molecule-Ig conjugate complex that gets taken up by cell
Conjugated radioisotope/cytokine/toxin exerts lethal effect

Question 19

Explain the use of proteases, pepsin and papain in the derivation of naturally derived antibody derivatives.

Answer 19

Both are involved in proteolytic cleavage.

Pepsin cleaves away the Fc domain to form F(ab)2

Papain cleaves away each Fab arm to form Fab

Question 20

Explain the structure of synthetically derived antibody derivatives: Bispecific and Triomabs

Answer 20

Bispecific: 2 distinct Fab regions to bind to 2 distinct epitopes, lack Fc domain

Triomabs: 2 distinct Fab region, presence of Fc domain to mediate Fc-dependent effector functions - CDC, ADCC (can bind to complement proteins and immune cells)

Question 21

Explain the structure of synthetic ScFv (Single chain variable fragment)

Answer 21

A single synthetic DNA fragment encodes for VH and VL in a single polypeptide chain (this includes the 6CDRs)

Question 22

Describe a naturally occurring fucosylated antibody.
What is its significance?

Answer 22

Fc domain is N-linked glycosylated (2 N-linked oligosaccharide chains bound to the Fc region). N-glycans attached to Asn297 in Fc domain are linked to fucose.
*Fucose is the glycan

Fucosylated Fc domain have reduced affinity to bind to FcyRIII receptor on effector cells, this reduces ADCC induction, therefore reduce efficacy of antibodies

Thus, defucosylated Abs (remove fucose from Asn297) have competitive edge as it has enhanced binding affinity to FcyRIII receptor, increase ADCC induction, incr efficacy